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Chemical Proteomics Methods and Protocols 1st Edition Marcus Bantscheff (Auth.) Chemical Proteomics Methods and Protocols 1st Edition Marcus Bantscheff (Auth.) Chemical Proteomics Methods and Protocols 1st Edition Marcus Bantscheff (Auth.)

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Chemical Proteomics Methods and Protocols 1st Edition Marcus Bantscheff (Auth.)
Chemical Proteomics Methods and Protocols 1st Edition Marcus Bantscheff (Auth.) Digital Instant Download Author(s): Marcus Bantscheff (auth.), Gerard Drewes, Marcus Bantscheff (eds.) ISBN(s): 9781617793646, 1617793647 Edition: 1 File Details: PDF, 4.02 MB Year: 2012 Language: english
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Chemical Proteomics Methods and Protocols 1st Edition Marcus Bantscheff (Auth.)
Chemical Proteomics Methods and Protocols Edited by Gerard Drewes and Marcus Bantscheff CellzomeAG,Heidelberg,Germany
Editors Dr. habil. Gerard Drewes Cellzome AG Heidelberg, Germany gerard.drewes@cellzome.com Dr. Marcus Bantscheff Cellzome AG Heidelberg, Germany marcus.bantscheff@cellzome.com ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-363-9 e-ISBN 978-1-61779-364-6 DOI 10.1007 /978-1-61779-364-6 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011937567 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or ­ dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)
v Preface An Overview of Chemical Proteomics: Methods and Applications The multidisciplinary science of chemical proteomics studies how small molecules of synthetic or natural origin bind to proteins and modulate their function. Scientists in the field have different backgrounds including molecular and cell biology, biochemistry, phar- macology, organic chemistry, and physics. Chemical Proteomics: Methods and Applications is directed at molecular biologists and biochemists with either an interest in small molecules themselves, e.g., in drug discovery projects, or in using small-molecule probes as research tools to study protein function. The book may also be useful for organic chemists with an interest in biology and for specialists in protein mass spectrometry. In the introductory chapters, we discuss analytical strategies for chemical proteomics projects, with a focus on the current state-of-the-art in protein mass spectrometry, and describe several examples how chemical proteomics can impact the field of drug discovery. The consecutive chapters provide detailed experimental protocols. Most chemical proteom- ics projects consist of three parts. In the first part, the chemical probe is selected or designed, and then synthesized. In the second part, the probe compound is exposed to the protein sample or cell extract. In the final part, proteins binding to the probe compound are identi- fied and often quantified. In simple applications, this is often achieved by antibody-based detection, but if the aim is the discovery of targets in an unbiased fashion, mass spectrom- etry is the method of choice. The following chapters cover all of these aspects. The first set of chapters describes how probes are generated from commercially available reagents without elaborate chemical synthesis procedures, and how the proteins binding to the probes can be analyzed by immunodetection or by mass spectrometry. Rix et al. and Saxena provide protocols for direct noncovalent affinity capture using protein kinase inhibi- tors as an example, which serves to profile the targets of these compounds and provides probes for kinase expression and activity. Ge and Sem have developed a target class-specific probe for the labeling of dehydrogenase enzymes. Kawamura and Mihai, and Codreanu et al. describe the use of biotin-conjugated probes which form covalent adducts with defined subproteomes, here consisting of adenine-binding proteins and targets of lipid electrophiles. Lenz et al. combine features of noncovalent and covalent capturing strategies in their bifunc- tional ligands designed to target methyltransferases, an emerging class of drug targets. The second set of chapters is concerned with techniques for the discovery of small- molecule targets and the probing of target function. Ong et al. describe the use of stable isotope labeling of amino acids in cell culture (SILAC) in identifying proteins that bind small-molecule probes in cell extracts. Hopf et al. perform affinity enrichment of target pro- teins on a probe matrix in the presence of competing free test compound in solution, thus enabling determination of binding potencies of the free test compound to affinity-captured proteins from cell extracts. The method employs quantitative mass spectrometry with iso- baric mass tags to determine the potencies for a large number of targets in a single analysis. Ge and Sem have developed a protocol for the detection and purification of dehydrogenase
vi Preface enzymes, which may represent targets, but also unwanted off-targets, for certain types of drugs. Kovanich et al. describe a combination of cAMP-based affinity chromatography with quantitative mass spectrometry to investigate protein kinase A complexes in extracts of cells and tissues. De Jong et al. use activity-based chemical probes to profile the activity of the proteasome, which has recently emerged as an important cancer target, in cells and tissues. The next three chapters provide innovative protocols for the study of potential drug targets by chemical cross-linking and mass spectrometry. Mueller et al. provide a method to study protein–drug interactions, and Gasilova et al. employ cross-linking and MALDI-mass spec- trometry to study ligand modulation of protein–protein interactions. Jeon et al. provide a protocol for in vivo cross-linking via time-controlled transcardiac perfusion, which in prin- ciple enables the direct analysis of protein targets in animal models. The final set of chapters is concerned with small-molecule ligand and drug discovery. Casalena et al. describe the discovery of probe compounds by utilizing compound libraries immobilized on microarrays. Wolf et al. delineate general guidelines for working with small molecules, including aspects like storage, the preparation of solutions, and the determina- tion of solubility. The chapter by de Matos et al. provides guidelines for the use of the ChEBI database, which should be very helpful for researchers tasked with the selection of a particular probe or with building a small molecule collection to purpose. They describe the Chemical Entities of Biological Interest (ChEBI) database which helps to find probe molecules with the desired structural or biological features. Finally, many researchers will consider whether their research tool compound might have the potential to be developed into a drug. Zhang delivers a lucid analysis of the features that distinguish drugs from probe molecules, and lays out a set of rules for “drug likeness.” Affinity- and activity-based chemical probes, combined with quantitative immunodetec- tion and mass spectrometry techniques, are increasingly gaining appreciation as powerful strategies for the molecular analysis of complex biological systems in homeostasis and dis- ease. We hope that the methodologies described in this volume will contribute to a wider application of chemical proteomics methods in biochemical and cell biological laboratories. Heidelberg, Germany Gerard Drewes Marcus Bantscheff
vii Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Part I Introduction 1 Mass Spectrometry-Based Chemoproteomic Approaches . . . . . . . . . . . . . . . . . . . . 3 Marcus Bantscheff 2 Chemical Proteomics in Drug Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Gerard Drewes Part II Small Molecules and Probe Design 3 Compound Immobilization and Drug-Affinity Chromatography . . . . . . . . . . . . . . 25 Uwe Rix, Manuela Gridling, and Giulio Superti-Furga 4 Affinity-Based Chemoproteomics with Small Molecule-Peptide Conjugates . . . . . . 39 Chaitanya Saxena 5 A Chemical Proteomic Probe for Detecting Dehydrogenases: Catechol Rhodanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Xia Ge and Daniel S. Sem 6 Probing Proteomes with Benzophenone Photoprobes . . . . . . . . . . . . . . . . . . . . . . 65 Akira Kawamura and Doina M. Mihai 7 Biotinylated Probes for the Analysis of Protein Modification by Electrophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Simona G. Codreanu, Hye-Young H. Kim, Ned A. Porter, and Daniel C. Liebler 8 Profiling of Methyltransferases and Other S-Adenosyl-l-Homocysteine- Binding Proteins by Capture Compound Mass Spectrometry . . . . . . . . . . . . . . . . 97 Thomas Lenz, Peter Poot, Elmar Weinhold, and Mathias Dreger Part III Target Discovery and Target Validation 9 Identifying Cellular Targets of Small-Molecule Probes and Drugs with Biochemical Enrichment and SILAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Shao-En Ong, Xiaoyu Li, Monica Schenone, Stuart L. Schreiber, and Steven A. Carr 10 Determination of Kinase Inhibitor Potencies in Cell Extracts by Competition Binding Assays and Isobaric Mass Tags . . . . . . . . . . . . . . . . . . . . 141 Carsten Hopf, Dirk Eberhard, Markus Boesche, Sonja Bastuck, Birgit Dümpelfeld, and Marcus Bantscheff 11 Affinity-Based Profiling of Dehydrogenase Subproteomes . . . . . . . . . . . . . . . . . . . 157 Xia Ge and Daniel S. Sem
viii Contents 12 Probing the Specificity of Protein–Protein Interactions by Quantitative Chemical Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Duangnapa Kovanich, Thin Thin Aye, Albert J.R. Heck, and Arjen Scholten 13 Fluorescence-Based Proteasome Activity Profiling . . . . . . . . . . . . . . . . . . . . . . . . . 183 Annemieke de Jong, Karianne G. Schuurman, Boris Rodenko, Huib Ovaa, and Celia R. Berkers 14 Chemical Cross-Linking and High-Resolution Mass Spectrometry to Study Protein–Drug Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 Mathias Q. Müller and Andrea Sinz 15 Monitoring Ligand Modulation of Protein–Protein Interactions by Chemical Cross-Linking and High-Mass MALDI Mass Spectrometry . . . . . . . . . . . . . . . . . . 219 Natalia Gasilova and Alexis Nazabal 16 Time-Controlled Transcardiac Perfusion Crosslinking for In Vivo Interactome Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 Amy Hye Won Jeon and Gerold Schmitt-Ulms Part IV Ligand Discovery 17 Ligand Discovery Using Small-Molecule Microarrays . . . . . . . . . . . . . . . . . . . . . . 249 Dominick E. Casalena, Dina Wassaf, and Angela N. Koehler 18 Working with Small Molecules: Preparing and Storing Stock Solutions and Determination of Kinetic Solubility . . . . . . . . . . . . . . . . . . . . . . . . . 265 Andrea Wolf, Satoko Shimamura, and Friedrich B.M. Reinhard 19 A Database for Chemical Proteomics: ChEBI . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Paula de Matos, Nico Adams, Janna Hastings, Pablo Moreno, and Christoph Steinbeck 20 Working with Small Molecules: Rules-of-Thumb of “Drug Likeness” . . . . . . . . . . . 297 Ming-Qiang Zhang Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
ix Contributors Nico Adams • Department of Genetics, University of Cambridge, Cambridge, UK Thin Thin Aye • Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University and Netherlands Proteomics Centre, Utrecht, The Netherlands Marcus Bantscheff • Cellzome AG, Heidelberg, Germany Sonja Bastuck • Cellzome AG, Heidelberg, Germany Celia R. Berkers • Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands Markus Boesche • Cellzome AG, Heidelberg, Germany Dominick E. Casalena • Chemical Biology Platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Steven A. Carr • Proteomics platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Simona G. Codreanu • Vanderbilt University School of Medicine, Nashville, TN, USA Annemieke de Jong • Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands Paula de Matos • European Bioinformatics Institute, Hinxton, UK Mathias Dreger • caprotec bioanalytics GmbH, Berlin, Germany Gerard Drewes • Cellzome AG, Heidelberg, Germany Birgit Dümpelfeld • Cellzome AG, Heidelberg, Germany Dirk Eberhard • Cellzome AG, Heidelberg, Germany Natalia Gasilova • CovalX AG, Schlieren, Switzerland; Department of Chemistry and Applied Biosciences, ETH, Zürich, Switzerland Xia Ge • Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI, USA Manuela Gridling • CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Janna Hastings • European Bioinformatics Institute, Hinxton, UK Albert J.R. Heck • Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University and Netherlands Proteomics Centre, Utrecht, The Netherlands Carsten Hopf • Cellzome AG, Heidelberg, Germany Amy Hye Won Jeon • Tanz Centre for Research in Neurodegenerative Diseases and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Akira Kawamura • Department of Chemistry, Hunter College of CUNY, New York, NY, USA Hye-Young H. Kim • Vanderbilt University School of Medicine, Nashville, TN, USA Angela N. Koehler • Chemical Biology Platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Duangnapa Kovanich • Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University and Netherlands Proteomics Centre, Utrecht, The Netherlands
x Contributors Thomas Lenz • caprotec bioanalytics GmbH, Berlin, Germany Xiaoyu Li • Chemical Biology platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Daniel C. Liebler • Vanderbilt University School of Medicine, Nashville, TN, USA Doina M. Mihai • Department of Chemistry, Hunter College of CUNY, New York, NY, USA Pablo Moreno • European Bioinformatics Institute, Hinxton, UK Mathias Q. Müller • Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany Alexis Nazabal • CovalX AG, Schlieren, Switzerland Shao-En Ong • Proteomics Platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Huib Ovaa • Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands Peter Poot • Institute of Organic Chemistry, RWTH University, Aachen, Germany Ned A. Porter • Vanderbilt University School of Medicine, Nashville, TN, USA Friedrich B.M. Reinhard • Cellzome AG, Heidelberg, Germany Uwe Rix • CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Boris Rodenko • Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands Chaitanya Saxena • Shantani Proteome Analytics Pvt. Ltd., Pune, MH, India Monica Schenone • Proteomics platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Gerold Schmitt-Ulms • Tanz Centre for Research in Neurodegenerative Diseases and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Arjen Scholten • Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University andNetherlands Proteomics Centre, Utrecht, The Netherlands Stuart L. Schreiber • Chemical Biology Platform Chemical Biology Program, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Karianne G. Schuurman • Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands Daniel S. Sem • Department of Pharmaceutical Sciences, Concordia University Wisconsin, Mequon, WI, USA Satoko Shimamura • Cellzome AG, Heidelberg, Germany Andrea Sinz • Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany Christoph Steinbeck • European Bioinformatics Institute, Hinxton, UK Giulio Superti-Furga • CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Dina Wassaf • Chemical Biology Platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA
xi Contributors Elmar Weinhold • Institute of Organic Chemistry, RWTH University, Aachen, Germany Andrea Wolf • Cellzome AG, Heidelberg, Germany Ming-Qiang Zhang • Merck Sharp Dohme (MSD), Chaoyang District, Beijing, PR, China
Chemical Proteomics Methods and Protocols 1st Edition Marcus Bantscheff (Auth.)
Part I Introduction
3 Gerard Drewes and Marcus Bantscheff (eds.), Chemical Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 803, DOI 10.1007/978-1-61779-364-6_1, © Springer Science+Business Media, LLC 2012 Chapter 1 Mass Spectrometry-Based Chemoproteomic Approaches Marcus Bantscheff Abstract The term “chemical proteomics” refers to a research area at the interface of chemistry, biochemistry, and cell biology that focuses on studying the mechanism of action of bioactive small molecule compounds, which comprises the mapping of their target proteins and their impact on protein expression and posttranslational modifications in target cells or tissues of interest on a proteome-wide level. For this purpose, a large arsenal of approaches has emerged in recent years, many of which employing quantitative mass spectrometry. This review briefly summarizes major experiment types employed in current chemical proteomics research. Key words: Affinity chromatography, Chemical proteomics, Drug profiling, Mass spectrometry, Posttranslational modifications, Quantitative proteomics, Stable isotope labeling, Targeted proteomics Experimental procedures commonly employed in chemical pro- teomics approaches can be categorized into two major groups (Fig. 1): (1) global proteomics approaches, aiming at the cell-wide characterization of cellular response to drug treatment, e.g., altered protein expression levels and posttranslational modifications; and (2) activity- or affinity-based protein profiling. The latter summa- rizing targeted chemoproteomic approaches which employ small molecular probes engineered to selectively capture protein targets/ subproteomes via a specific binding mode (e.g., by targeting co- factor binding sites) (1–8). 1. Introduction
4 M. Bantscheff For global chemoproteomic profiling, cells or animals are treated with a drug before system-wide proteome analysis to evaluate the cellular response in a global way (9–12). This strategy has become very attractive because of its simplicity and the unbiased nature of the analysis. Apart from cell permeability, there are no particular requirements for the small molecule compounds to be tested in such assays and the required quantitative mass spectrometric tech- niques are now available in many laboratories (for recent reviews, see refs. 13–16). However, since typically no protein enrichment is used, the changes that can be observed are limited by the analytical depth of the analysis and are often restricted to more abundant proteins. The analysis is further complicated by the fact that pro- teins found to be altered in abundance are not necessarily direct targets or downstream of the affected signaling cascade but often represent highly abundant proteins involved in stress response and/or housekeeping functions (12, 17). Hence, it is almost never possible to distinguish direct drug protein interactions from indi- rect effects. In examples illustrating the advantages and limitations 2. Global Chemoproteomic Profiling a b Wash Digest proteins LC-MS/MS Extract proteins Add Beads m/z Purify probes on beads Wash Digest proteins LC-MS/MS Extract proteins Add probe React probe c m/z Digest proteins LC-MS/MS Extract proteins m/z +Compound Control +Compound Control +Compound Control m/z m/z m/z 1 a.i. 1 a.i. 1 a.i. 1 a.i. 1 a.i. 1 a.i. Fig. 1. Experimental workflows in chemical proteomics. (a) Global proteomics approaches: Cells are treated with a com- pound, proteins are extracted from the sample, digested to peptides and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). (b) Activity-based protein profiling (ABPP) utilizes reactive probes specifically targeting the active site of an enzyme family. After protein extraction, the lysate is incubated with the probe to covalently attach to its targets. In the second step, probes and targets are purified using affinity chromatography before digestion and LC-MS/MS analysis. Pretreatment of cells with a small molecule compound binding to the active site of the investigated enzyme family leads to reduced capturing of the target enzyme via the reactive probes. (c) Alternatively, the compounds of interest can be modified and immobilized on a solid support. The immobilized drug is subsequently incubated with a cell extract to specifically enrich for target proteins that are subsequently identified by mass spectrometry. Competition with free excess inhibitor reduces the abundance of captured target proteins.
5 1 Mass Spectrometry-Based Chemoproteomic Approaches of global proteomics approaches, Chen and co-workers (12, 18, 19) evaluated the differential effect of the R- and S-enantiomers of atenolol, a β1-selective adrenoreceptor blocker, and the nonsteroi- dal antiinflammatory drug ibuprofen on two different cell types. The authors found 27 and 13 proteins to be differentially expressed, most of which can be classified as highly abundant (17). Yamanaka and co-workers applied a global proteomics approach for toxico- logical studies in animals (20). The authors studied the effects of 63 chemical compounds on protein expression in rat liver after 28 daily dosings and employed statistical methods to detect proteins characteristic for carcinogenicity. Subcellular and chromatographic fractionation allow for a more directed analysis of drug-induced changes in protein expression or posttranslational modifications. Lee and co-authors monitored his- tone modifications in response to treatment with histone deacety- lase (HDAC) inhibitors (10). In this study, human colon cancer cells were treated with HDAC inhibitors of varying degrees of selectivity followed by a simple prefractionation method to enrich for histone proteins. By employing label-free quantitative mass spectrometry, the authors identified HDAC-dependent histone acetylation patterns and quantified these in response to inhibitor treatment. Similarly, JmjD2A-dependent demethylation of K9 in histone H3 was monitored in response to cell treatment with pyridine-2,4-dicarboxylic acid derivatives (21). In several recent studies, chromatographic- or antibody-based enrichment methods have been employed to analyze kinase inhibitor-induced changes in protein phosphorylation on a global scale, thus mapping the net- work-level response to inhibitor treatment, and to infer signaling network topology (2, 11, 22, 23). A study by Mann and co-workers illustrated the impressive analytical depth that has been achieved in phosphoproteomics (22). Triple labeling SILAC (stable isotope labeling by amino acids in cell culture (24)) was used to analyze phosphorylation levels in growth factor-stimulated cells in the pres- ence or absence of kinase inhibitors. Among thousands of phospho- peptides, fewer than 10% were affected by the MAPK inhibitors U0126 and SB202190. By contrast, almost 1,000 phosphopeptides were affected by treatment of the leukemia cell line K562 with the potent but unselective BCR-Abl inhibitor dasatinib. A similar approach was applied to quantify changes in protein acetylation in response to the deacetylase inhibitors SAHA and MS-275 (25). In contrast to global approaches, recent developments in affinity- based proteomics techniques have enabled to directly determine protein binding profiles of small molecule drugs under close-to physiological conditions. These techniques are in principle based 3. Targeted Chemoproteomics Approaches
6 M. Bantscheff on affinity chromatography, typically using immobilized drugs or tool compounds (1, 8, 26) or covalent active site-labeling probes (3, 6, 27). Activity-based protein profiling (ABPP) via reactive probes designed to specifically bind to active sites of target enzymes was pioneered by the Cravatt laboratory. In this approach, the reactive probe is typically fused to an affinity tag, such as biotin, via a spacer. In a first step, the small molecule probe is incubated with the bio- logical sample and allowed to covalently attach to proteins it has affinity for. Subsequently, the formed probe–protein conjugates are captured using the affinity tag. Probes have been developed for a variety of enzyme classes including hydrolases (28), proteases (29, 30), kinases (31, 32), phosphatases (33), histone deacetylases (34), and glycosidases (35). For example, fluorescent activity-based probes were reported that enable substrate-free identification of inhibitors of uncharacterized enzymes. By using fluorescence polarization as a read-out, the approach is compatible to high- throughput screening with recombinant enzymes (36). A recent report demonstrated how ABBP can be used to screen compound libraries against an entire target class (37). The authors first synthe- sized a probe to selectively capture 80% of mammalian serine hydrolases, then screened 70 SHs against 140 structurally diverse carbamates and assessed the selectivity of hits using the very same approach. Patricelli and co-workers (31) used acyl phosphate- containing nucleotides, prepared from a biotin derivative and ATP or ADP to covalently modify ATP-binding proteins directly in cell extracts. Activity-based probes can be adapted for in situ and in vivo labeling by introducing a bio-orthogonal chemical handle, such as an alkyne. Probe-labeled enzymes are then captured by click chem- istry conjugation to azide-containing reporter tags (38–40). Probe design is a crucial step when developing ABPP-based assays and often requires detailed structural information to ensure selective binding of the probe to the catalytic site of target enzymes and to enable the efficient reaction of the active probe with suitable amino acid residues within or close to the catalytic site. In order to streamline probe development, a trifunctional probe design has been suggested consisting of a reversibly interacting selectivity group, a reactive group, and a sorting function (41). In this strategy, small molecules generating selectivity for individual enzyme classes are attached to a common building block containing reactive group and sorting function (i.e., biotin) that is common to all probes. Recent reports demonstrated examples for profiling of cAMP-binding pro- teins (42), kinases (43), and methyltransferases (44). For probes interacting with target enzymes with high affinity, however, probe design can be drastically simplified by omitting the reactive group and directly immobilizing the compound of interest on a solid support. The thus generated probe matrix is then incu- bated with cell extracts and mass spectrometry can be employed in
7 1 Mass Spectrometry-Based Chemoproteomic Approaches order to identify capture proteins (5, 45). Recent reports demonstrated successful applications of this approach for enrichment of protein kinases and characterization of binding profiles of kinase inhibitors (13, 23, 46–51), proteins binding to ATP/ADP (52), phosphati- dylinositols (53, 54), cyclic nucleotides (55, 56), histone deacety- lases (57), and peptides (58–60). Further, in several recent mode-of-action studies, immobilized analogs of lead compounds enabled target identification (61–64). Several large-scale studies have been performed for target class specific enrichment of kinase inhibitors using both reactive probes targeting the ATP binding pocket (31, 43) and immobilized unselective kinase inhibitors (46, 65) for affinity enrichment. The kinome coverage achieved with kinase inhibitor probes compared quite favorably to results obtained with reactive probes, indicating a limited impact of the bond-forming reaction with kinases on target class coverage. Qualitative binding profiles obtained in simple activity/affinity enrichment studies give only limited information about binding potencies of targets and off-targets detected. Consequently, the pharmacological relevance of detected off-target protein has to be validated using the standard repertoire of enzymatic assays (13, 47, 49, 50). While this might be feasible for very selective probes, cap- turing only few proteins, with recent mass spectrometric equip- ment often hundreds of proteins can be identified from a single affinity enrichment experiment. Further, the analysis of complex mass spectrometry data is impaired by the fact that the amounts of individual proteins captured do not represent the affinities of these proteins to the immobilized compound. Hence, additional evi- dence is required to distinguish low-abundant high-affinity inter- actors from low-affinity abundant ones, e.g., albumin and hemoglobin are known to have low affinity for a range of small molecules and many NADH/NADPH binding proteins bind to immobilized ATP-mimetics (13, 47, 49–51). In addition, proteins might bind to the resin or additional groups introduced to com- pounds for probe generation, e.g., linkers, reactive groups, biotin, etc. Some of these issues can be addressed by excluding those pro- teins from further analysis that have been frequently observed in independent experiments using different probe matrices (66). An elegant way to increase over-all specificity of the experiment is to design two probes/probe matrices, one containing the active com- pound of interest and one containing an inactive analog (45). Experiments with both matrices are then performed in parallel and candidate target proteins can be shortlisted upon differential display of results, e.g., using quantitative mass spectrometric methods. 4. Impact of Experiment Design and Quantitative Read-Out for Target Identification
8 M. Bantscheff However, inactive analogs are often not available and designing two independent probes is quite laborious. Utilizing reactive or immobilized analogs of a small molecule compound in order to identify its targets is a rather indirect approach and modifications introduced for probe generation might alter potency and selectivity of the compound under investigation. This limitation can be addressed by a competition-based experi- ment design. In this approach, probe/probe matrix binding is per- formed in the presence or absence of an excess of free, underivatized compound. Experiments are performed in parallel and quantitative mass spectrometry can be applied to identify those proteins for which captured amounts are strongly reduced in the presence of free compound as compared to experiments performed with vehi- cle control. In a recent application of this approach, Borawski et al. generated an affinity matrix consisting of a derivatized, bioactive PI4KB inhibitor linked to Sepharose beads used to purify cellular and/or viral proteins from a Huh7 HCV replicon cell lysate (61). To determine and quantify specific binding, the experiment was performed in a competition format by adding 10 μM PIK4B inhib- itor or DMSO alone into the replicon cell lysate prior to affinity purification. Bound proteins were eluted, digested with trypsin, and labeled with isobaric mass tags for relative and absolute quan- tification (iTRAQ, (67)) and combined prior to LC-MS/MS anal- ysis. This enabled to quantify binding displacement in the presence of free inhibitor relative to the vehicle. Several hundred proteins were identified and quantified in this experiment but only the bind- ing of class III PI4 kinases, PI4KA and PI4KB, was significantly reduced in the presence of free inhibitor. A similar, SILAC-based strategy was described by Ong et al. for the identification of targets of kinase inhibitors and imunophilin binders (68). Haystead and co-workers suggested a competition-based approach to estimate relative potencies of target proteins. ATP was linked to Sepharose-beads through the gamma phosphate group, thus generating a probe matrix selective for ATP-binding proteins including protein kinases as well as a variety of other proteins uti- lizing purine co-factors (52). After enrichment of ATP-binding proteins from cell extracts, the matrix was incubated with increas- ing concentrations of a set of antimalarial compounds and target proteins were eluted in a dose-dependent manner. In a variation of this approach, Patricelli and co-workers (31) used acyl phosphate- containing nucleotides, prepared from a biotin derivative and ATP or ADP to covalently modify ATP-binding proteins in the co-factor binding site. Biotinylated peptide fragments from labeled pro- teomes were subsequently captured and identified by mass spectrometry. Target kinases of kinase inhibitors were then deter- mined by comparing MS signals of probe-captured kinases in the absence and presence of excess free kinase inhibitors.
9 1 Mass Spectrometry-Based Chemoproteomic Approaches Stable isotope labeling-based quantitative mass spectrometry techniques for precise and accurate relative quantification of proteins (14, 16) have become an indispensable tool in chemical proteomic experiments, since identification of target proteins and their bind- ing affinities (IC50 s) largely depends on the ability to quantify differ- ences between vehicle control samples and samples incubated with different amounts of inhibitor. For example, iTRAQ labeling and LC-MS/MS were combined with a mixed kinase inhibitor probe matrix (“kinobeads”) for selectivity profiling of three inhibitors of the tyrosine kinase ABL developed for the treatment of chronic myeologeneous leukemia (CML); the phase II compound SKI-606 and the marketed drugs imatinib (Glivec) and dasatinib (46). In this study, cells or cell extracts were treated with inhibitor com- pounds at varying concentrations followed by incubation with the mixed kinase inhibitor matrix. Kinase inhibitors blocked the ATP binding pockets of target and off-target proteins as a function of concentration and affinity and consequently caused reduced abun- dance of these target proteins on the kinobeads matrix. Quantitative mass spectrometric analyses revealed binding profiles comprising 500 proteins including ~150 kinases. While dasatinib and SKI- 606 revealed very broad target profiles (46 and 42 proteins, respec- tively, showed 50% competition at 1 μM), imatinib was much more selective. In addition to the primary imatinib targets ABL/ BCR-ABL, ARG two novel target candidates, the receptor tyrosine kinase DDR1 (90 nM), and the quinone oxidoreductase NQO2 (43 nM) were identified and validated in independent studies (69, 70). Similarly, SILAC (24) has been employed in conjunction with kinase inhibitor matrices in order to determine the target profile of gefitinib (71). In a very recent report, such a chemoproteomics approach was applied to the analysis of inhibitors binding to native megadalton HDAC complexes. A total of 16 structurally diverse HDAC inhibitors were characterized for their selectivity in target- ing multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles, with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex, thus suggesting that the selectivity of HDAC inhibitors should be evaluated in the context of HDAC complexes rather than purified catalytic subunits (57). When cells are treated with an inhibitor, direct targets will be revealed by their reduced binding to the affinity matrix, however, e.g., protein kinases downstream of the respective target kinases will display an altered phosphorylation state due to the reduced signaling by the target kinase. For example, the case of imatinib- treated K562 cells, RSK1, and RSK3 were prominent examples for proteins exhibiting a significant downregulated phosphorylation state (46, 72).
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15 Gerard Drewes and Marcus Bantscheff (eds.), Chemical Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 803, DOI 10.1007/978-1-61779-364-6_2, © Springer Science+Business Media, LLC 2012 Chapter 2 Chemical Proteomics in Drug Discovery Gerard Drewes Abstract Real-world drug discovery and development remains a notoriously unproductive and increasingly uneconomical process even in the Omics era. The dominating paradigm in the industry continues to be target-based drug design, with an increased perception of the role of signaling pathways in homeostasis and in disease. Since proteins represent the major type of drug targets, proteomics-based approaches, which study proteins under relatively physiological conditions, have great potential if they can be reduced to practice such that they successfully complement the arsenal of drug discovery techniques. This chapter discusses examples of drug discovery processes where chemical proteomics-based assays using native endogenous proteins should have substantial impact. Key words: Chemical Proteomics, Drug target, Target deconvolution, Target validation, Drug discovery, Selectivity profiling Despite the dawn of the Omics era, drug discovery and develop- ment remains a notoriously unproductive and increasingly uneco- nomical process (1, 2). The dominating paradigm in the industry is still target-based drug design, with an increased perception of the role of signaling pathways in homeostasis and in disease (3). Because proteins represent the major type of drug targets, pro- teomics-based approaches, which allow to study a wide variety of proteins under relatively physiological conditions, have great potential if they can be reduced to practice such that they success- fully complement the arsenal of drug discovery techniques (4). Industry standard assays of drug action typically assess the biochemical activity of the purified target protein in isolation. Frequently, recombinant enzymes or protein fragments are used instead of the full-length endogenous proteins. The activity of a 1. Introduction
16 G. Drewes compound determined in this type of assays is often not predictive for its pharmacodynamic efficacy. One reason for this discrepancy is that an isolated recombinant protein, or protein fragment, does not necessarily reflect the native conformation and activity of the target in its physiological context, because of the lack of regulatory domains, expression of alternative splice variants, interacting regu- latory proteins, or incorrect protein folding or posttranslational modifications. As a consequence, data generated in such assays may not be predictive for the effects of a compound or drug in cell- based or in vivo models. Ideally, assays should be developed to generate data on native proteins in cell extracts or cell fractions, under conditions carefully optimized to preserve protein integrity, folding, posttranslational modification state, and interactions with other proteins. Both activity-based and affinity-based chemical proteomics techniques, as described in this volume, should com- plement, or in some instances replace the traditional recombinant protein-based assays. In target-based drug discovery, a project begins with the nomina- tion of a target. The target is typically defined as a protein which should be 1. Tractable: Its biochemical activity can be modulated by the desired therapeutic agent (e.g., a small molecule) in a dose- dependent fashion. 2. Validated: It mediates a pathophysiological process such that its modulation reverses a disease-relevant parameter, which can be measured in disease-related cell-based or animal models, and is expected to be predictive of human disease. Targets are often referred to as “druggable” and “clinically validated” when the modulation of the target was demonstrated to lead to the desired clinical outcome. Historically almost all drug- gable targets belong to a small number of target classes, biased toward cell surface proteins (e.g., G protein-coupled receptors, ion channels, or transporters) and a small number of intracellular protein classes (e.g., nuclear receptors, metabolic enzymes, kinases, or phosphodiesterases). A recent study estimated that the entirety of approved small molecule drugs acts through approximately 200 human proteins as targets (5), obviously a small number when compared to the 20,000–25,000 protein-coding genes in the human genome (6). It has been estimated that ten times as many suitable drug targets may exist, waiting to be discovered (7). In fact there are numerous proteins in pathways with a strong disease 2. Chemical Proteomics Can Aid More Informed Selection and Validation of Targets
17 2 Chemical Proteomics in Drug Discovery implication, e.g., based on pathobiochemical and human genetic evidence, which are not tractable by current small molecule-based approaches. Chemical proteomics approaches should serve to expand the number of accessible drug targets by aiding the identi- fication of tractable targets without the heavy bias toward the traditional target classes. This type of “target deconvolution” approaches was pioneered by the Schreiber laboratory in the clas- sical studies which identified the molecular targets of immunosup- pressants (8, 9). More recent exemplary approaches employed a combination of screening of diverse compound libraries in cell- based assays, which are not biased toward a particular family of targets, with chemoproteomics-based target identification. Huang et al. discovered the tankyrase proteins as tractable targets in the Wnt signaling pathway, which plays a central role in colon cancer but was characterized by a dearth of tractable drug targets (10). Using a related strategy, Fleischer et al. found that the potent and selective cytotoxic agent CB30865 exerts its effects by inhibition of nicotinamide phosphoribosyltransferase, an enzyme in the NAD biosynthetic pathway which helps cancer cells to sustain their increased energy metabolism (11). In another recent study, cell- based screening was performed for the upregulation of apolipopro- tein AI production, and the proteomic profiling of hit compounds led to the unexpected discovery of bromodomain proteins as tractable targets for the modulation of the expression of apolipo- protein AI and certain proinflammatory genes (12). These bro- modomain inhibitors exhibit a novel mechanism of action by blocking a protein–protein interaction formed between acetylated histones and BET-family bromodomains, which were not previ- ously regarded as tractable targets. These and other successful studies support the notion that there is a general need for small molecules as research tools to study protein function, particularly for proteins which are not classical drug targets. Both the Structural Genomics Consortium (http:/ /www.thesgc.org) and the Center for Protein Research (http:/ /www.cpr.ku.dk) have recently initi- ated extensive programs for the development of chemical probes which will be made available to the scientific community. Many drug discovery assays rely on the ability to express and purify the target protein in active form in the substantial amounts – typically milligrams of pure protein – necessary for the screening of com- pound libraries. The drug industry has encountered many so-called “difficult” target classes where this is not easily achieved, for instance, because the target protein is very large or requires addi- tional factors like interacting proteins for proper activity. Therefore, 3. Chemical Proteomics-Based Screening of Compound Libraries
18 G. Drewes methods based on immobilized probe compounds to capture the target directly from a cell or tissue extract without further purifica- tion can represent a viable alternative strategy. This approach was used by Fadden et al. who captured purine-binding proteins from porcine tissue with ATP-derivatized Sepharose and performed affinity elutions with 5,000 different compounds, resulting in the identification of 463 small molecule compounds eluting a total of 77 distinct proteins. Among these, novel and structurally diverse inhibitors of the cancer target Hsp90 were identified, which were further optimized to enter clinical development (13). A different strategy was used by Bantscheff et al. who screened a compound library for histone deacetylase (HDAC) inhibitors in a human cell line extract, using an immobilized hydroxamate-based probe. Here, compounds were added directly to the cell extract rather than using them for elution, such that each compound was assayed for the inhibition of the binding of HDACs to the immobilized probe (14). An important feature of both approaches is that the entire complement of proteins binding selectively to the immobi- lized probe is screened simultaneously. This represents a major advantage over traditional screening approaches, in particular, for target classes with a substantial number of structurally related tar- gets, like protein kinases or deacetylases, because possible “off- targets” (undesired additional proteins, which typically share a related active site with the target) are revealed early in the project. In conventional approaches, one is left to resort to educated guesses regarding possible “off-targets,” and distinct assays have to be con- figured for each individual protein. Despite the fact that drugs are usually optimized against a single target, many compounds exhibit polypharmacology, i.e., they act on multiple targets. These “off-targets” can increase the therapeu- tic potential of a drug, but they might also cause toxic side effects, which represent a major reason why drugs fail in clinical develop- ment (15). An important recent example was the chemoproteomics- based identification of cereblon (CRBN) as a target of the drug thalidomide which mediates the drug’s teratogenic effects (16). However, for oncology drugs, polypharmacology is the rule rather than the exception, as they often target proteins from large target classes with a high degree of structural conservation around the active site, like protein and lipid kinases, HDACs, or heat shock proteins. Compared to a truly selective drug, such a spectrum of targets is more likely to produce toxic side effects, but in oncology the increase in therapeutic potential may outweigh this disadvan- tage (17). Conventional strategies typically rely on assay panels comprising 10–100 purified enzymes to address compound 4. Chemical Proteomics for Drug Target Profiling
19 2 Chemical Proteomics in Drug Discovery potency, selectivity, and potential off-target liabilities (18). The recent progress in affinity-based proteomic techniques has enabled the direct determination of protein-binding profiles of small mol- ecule drugs under close to physiological conditions. These tech- niques utilize immobilized compounds as noncovalent affinity baits (14, 19–22) or covalent active-site labeling probes (23, 24). The affinity probes are designed to selectively enrich a larger set of up to several hundreds of proteins defined by structurally related active sites, which can be viewed as chemically tractable subproteomes (25). Noncovalent probes are used either immobilized to an affin- ity matrix like sepharose or conjugated to biotin, and have been used successfully for purine-binding proteins (26), protein kinases including transmembrane receptor kinases (21, 22), lipid kinases (27, 28), phosphodiesterases (29), and HDACs (14). Covalent active-site labeling probes are typically biotin conjugates and have been applied to kinases (30), GTPases (31), methylases (32), dehy- drogenases (33), serine-, cysteine-, metallo-, and proteasomal pro- teases (23, 34, 35), and HDACs (36). These methodologies typically generate protein affinity profiles for the immobilized com- pounds, which may reveal novel target candidates, but precautions must be taken to avoid false positives due to the background prob- lems caused by nonspecific interactions with abundant proteins. Moreover, for the application to drug discovery, e.g., in screening or affinity/selectivity profiling assays, the generation of robust quantitative data for hit and lead compounds is an absolute neces- sity. These problems can be managed if the affinity capture proto- cols are formatted as quantitative competition-binding assays. This can be achieved by adding the compound of interest in its free form in the tissue extract, before or together with the affinity matrix or the active site label, such that the free compound binds to its targets in the lysate, thereby effectively competing with the capturing probe. By assaying the free compound in the cell extract over a range of concentrations, dose–response binding curves are generated for as many proteins as can be captured by the probe compound and robustly quantified. In case of the “Kinobeads” matrix for protein kinases and the hydroxamate matrix for HDACs developed by Bantscheff et al., more than 1,000 proteins were found to bind to the matrix and were routinely quantified in drug- profiling experiments using a competition binding assay format coupled to protein quantification by isobaric tagging and high- resolution LC-MS/MS peptide sequencing (14, 22). For a more detailed discussion of qualitative and quantitative small molecule target profiling, the reader is referred to recent comprehensive reviews (20, 23, 37). Finally, in addition to the in vitro applications described above, many chemical proteomics strategies can poten- tially be adapted to the identification and activity profiling of tar- gets in living cells and in animal models (38). In conclusion, the recent advances in chemical proteomics and in analytical instrumentation have promoted new drug discovery
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Part II Small Molecules and Probe Design
25 Gerard Drewes and Marcus Bantscheff (eds.), Chemical Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 803, DOI 10.1007/978-1-61779-364-6_3, © Springer Science+Business Media, LLC 2012 Chapter 3 Compound Immobilization and Drug-Affinity Chromatography Uwe Rix, Manuela Gridling, and Giulio Superti-Furga Abstract Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to under- standing its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identifica- tion of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobiliza- tion, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatogra- phy step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry. Key words: Drug, Target, Affinity chromatography, Immobilization, Chemical proteomics, Mechanism of action Promiscuity of small molecule drugs and tool compounds has important implications for understanding their cellular mecha- nisms, side effects, and clinical potential. Chemical proteomics is a postgenomic, mass spectrometry (MS) and bioinformatics-enabled version of previous versions of drug-affinity chromatography that can identify a drug’s cellular target profile, thus aiding in the subsequent 1. Introduction
26 U. Rix et al. characterization of its molecular mechanism of action (1, 2). This approach requires the immobilization of the compound of interest on solid support as an early step. The strategy for immobilization has to be designed very carefully as chemical modification of any bioactive compound harbors the risk of impairing or abrogating its biological activity through disruption of the protein interaction interface. Therefore, one should utilize all available structure– activity relationship information, which can, for instance, be obtained by activity studies with structural analogs or from X-ray co-crystal structures. If such information is unavailable, it is recom- mended to immobilize the bait compound in separate experiments via two or more different linker attachment points. In some cases, when a cellular or biochemical readout is at hand, these analogs can be tested for retention of activity. When employing cellular assays, though, it has to be kept in mind that modifying a compound might also alter its cell permeability properties. In addition, some molecules are sensitive to various commonly employed conditions, such as acid, base, or heat. Therefore, any broadly applicable modification or immobilization reaction has to be efficient and mild. This is the case for several different reactions employing, e.g., epoxide or amide chemistry (3). The formation of an amide bond from an activated N-hydroxy-succinimide (NHS) ester and the amino group (primary or secondary) of a bioactive compound (analog) is readily achieved at room temperature and the reaction usually comes to completion within several hours (4). Compounds containing hydroxy- or carboxy-groups can be conveniently converted into amines by Steglich esterification with Boc- or Fmoc-protected amino acids or mono-protected ethylenediamine, respectively, and subsequent deprotection (5). The choice of protecting group should be based on chemical stability of the bait compound under deprotection conditions (6). Other important considerations for drug-affinity chromatography are the choice of buffer and pH. Just as critical is the efficient inhibition of cellular proteases, which would destroy proteins and prevent affinity enrich- ment, and possibly enzymes that carry out unscheduled posttrans- lational modifications in vitro (e.g., protein dephosphorylation), as well as the choice and concentration of detergent, which aids in the solubilization of proteins. In the following, we present the detailed workflow for immobilization and affinity chromatography, which we have previously applied to the characterization of several clinical kinase inhibitors by chemical proteomics (7). We exemplify the approach with a study on the BCR-ABL tyrosine kinase inhibitor dasatinib (BMS-354825, trade name Sprycel) (8), a drug in clini- cal use for chronic myeloid leukemia (CML) and several other malignancies. In this example, drug-affinity chromatography is performed using the CML cell line KU812 (9).
27 3 Compound Immobilization and Drug-Affinity Chromatography 1. NHS-activated Sepharose 4 Fast Flow (GE Healthcare) (see Note 1). 2. Dimethyl sulfoxide, absolute over molecular sieve (DMSO, Fluka). 3. Isopropanol, absolute over molecular sieve (iProp, Fluka). 4. Triethylamine (TEA, Sigma). 5. Ethanolamine (Aldrich). 6. 2 mL microcentrifuge tubes (Eppendorf). 1. Methanol (MeOH), LC-MS grade (Fisher Scientific GmbH). 2. Isopropanol (iProp), LC-MS grade (Fisher Scientific GmbH). 3. Water, LiChrosolv grade (Merck). 4. Formic acid. 5. Chromatography column: SunFire C18 , 5 μm, 3.0×50 mm (Waters). 6. HPLC 2795 (Waters). 7. Photo diode array detector 2996 (Waters). 8. ZQ 2000 single quadrupole mass spectrometer (Waters/ Micromass). 1. Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) sup- plemented with 10% fetal bovine serum (Gibco) and penicillin/ streptomycin 100× (PAA Laboratories GmbH). 2. Cell culture dishes, Ø 150 mm (VWR International). 3. 4× Concentrated Laemmli sample buffer containing 10% β-mercaptoethanol. 4. Lysis buffer 1×: 50 mM Tris–HCl, 100 mM NaCl, 0.2% NP-40, 5% glycerol, 1.5 mM MgCl2 , 25 mM NaF, 1 mM Na3 VO4 , 1 mM phenylmethylsulfonyl fluoride, 1 mM dithio- threitol (DTT), 10 μg/mL TLCK, 1 μg/mL leupeptin, 1 μg/ mL aprotinin, and 10 μg/mL soybean trypsin inhibitor (Sigma), pH 7.5 (see Note 2). 5. γ-Globin (1 μg/μL) (Bio-Rad). 6. Protein Assay Dye Reagent Concentrate (5×) (Bio-Rad). 7. Hypodermic Needle, 20 G×1½ (Terumo® ), single-use syringe 10 mL (B. Braun Melsungen AG). 8. Cuvettes half-micro, 2.5 mL, PMMA (VWR International). 9. Polycarbonate ultracentrifuge tubes (Beckman Coulter). 10. Ultracentrifuge and fixed-angle rotor (Beckman Coulter). 2. Materials 2.1. Coupling to Solid Support 2.2. LC-MS Analysis of the Coupling Reaction 2.3. Cell Culture, Harvest, Lysis, and Protein Quantification
28 U. Rix et al. 1. Lysis buffer 1× (see above). 2. 4× Concentrated Laemmli sample buffer (containing 10% β-mercaptoethanol) (see above). 3. Polycarbonate ultracentrifuge tubes (Beckman Coulter). 4. Spin columns Mobicol (MoBiTec GmbH). 5. Spin column lower filters – 90 μm pore size (MoBiTec GmbH). 6. 1.5 and 2 mL microcentrifuge tubes (Eppendorf). 1. Iodoacetamide (13 μg/μL) (Sigma). 2. Mini-PROTEAN Tetra electrophoresis system (Bio-Rad Laboratories). 3. 4× Running gel buffer (SDS buffer): 1.5 mM Tris–HCl and 0.54% SDS, pH 8.8. 4. 4× Gel buffer 2: 2 M Tris, 1.6 mM SDS, pH 6.8. 5. Ammoniumperoxide sulfate (APS), acrylamide, tetramethyl- ethylenediamine (TEMED, Sigma). 6. 5× Running buffer: 602 g Tris, 2,880 g glycine, 200 g SDS, 20 L water, pH 8.3. Do not use HCl! 7. Broad range 7–175 kDa prestained protein marker (New England Biolabs). 1. Fixing buffer: 40% ethanol, 10% acetic acid. 2. Washing buffer: 35% ethanol or distilled water. 3. Sensitizing buffer: 0.02% Na2 S2 O3 . 4. 0.2% AgNO3 solution (refrigerated). 5. Developing buffer: 3% Na2 CO3 , 0.05% formaldehyde. 6. Quenching solution: 5% acetic acid. 1. 10× Western blot buffer: for 2 L dissolve 60.7 g Tris and 288.4 g glycine in distilled water. 2. 1× Western blot buffer: 10% (v/v) 10× Western blot buffer, 10% (v/v) methanol. 3. Wash buffer: PBS with 1% Tween-20 (PBS-T). 4. Blocking buffer (in this example, see Note 3): 3% bovine serum albumin (BSA) in PBS-T. 5. Primary antibody dilution (in this example): 1:2,000 mono- clonal mouse anti-ABL (21–63) (Santa Cruz Biotechnology) in 3% BSA/PBS-T (see Note 4). 6. Secondary antibody dilution (in this example): 1:2,000 horse radish peroxidase (HRP)-conjugated anti-mouse (Rockland Immunochemicals) in PBS-T. 2.4. Affinity Pulldown 2.5. One-Dimensional SDS-PAGE 2.6. Silver Staining 2.7. Immunoblot
29 3 Compound Immobilization and Drug-Affinity Chromatography 7. Amersham ECL Plus™ Western Blotting Detection Reagents (GE Healthcare). 8. Nitrocellulose transfer membrane (Protran BA 85, 300 mm×3 m, 0.45 μm). 9. Saran wrap. 10. Amersham Hyperfilm ECL (GE Healthcare). 11. Hoefer Semi-Phor semi-dry transfer unit (Hoefer, Inc.). This section is illustrated in Fig. 1. 1. The beads are provided in a slurry of approximately 50% iso- propanol. For one pull down, pipette 100 μL of this slurry into a 1.5 mL microcentrifuge tube (see Note 5). Compare the settled bed volume with 50 μL DMSO. Add or remove slurry until 50 μL bed volume is reached (see Note 6). 3. Methods 3.1. Coupling to Solid Support Fig. 1. Immobilization strategy for the BCR-ABL kinase inhibitor dasatinib. The “couple- able” analog c-dasatinib was designed based on the X-ray co-crystal structure of ABL with dasatinib, which indicated that the hydroxy group extends into the solvent space (10). The dasatinib affinity matrix is generated via coupling of c-dasatinib to NHS-activated sepharose.
30 U. Rix et al. 2. Centrifuge the beads for 3 min at room temperature with 75×g and remove the supernatant. 3. Add 50 μL of DMSO, resuspend gently by inverting several times, centrifuge (as before), and discard the supernatant. Repeat this washing step three times with 0.5 mL DMSO. Then resuspend beads in 50 μL DMSO. 4. Dissolve bait compound to a final concentration of 10 mM in DMSO and add 5 μL of this stock solution and 0.75 μL TEA to the 50% bead slurry, mix carefully and incubate on roto- shaker for 16–24 h (but at least for 8 h) at room temperature with 10 rpm (see Note 7). 5. Centrifuge the beads (as before) and remove 10 μL (»5 nmol) of the supernatant for the LC-MS control. This is the T16 time point for the coupling control. 6. If the drug was successfully coupled to the beads, block the unreacted NHS-ester groups by adding 25 μL ethanolamine and incubate on roto-shaker for further 16–24 h (but at least for 8 h) at room temperature with 10 rpm. 7. Centrifuge beads, remove the supernatant, wash twice with 0.5 mL of DMSO, and discard the supernatant again. 8. Either proceed directly with the drug pulldown and wash the beads with Lysis buffer or add 50 μL isopropanol, centrifuge, discard the supernatant, and repeat this washing step twice with 0.5 mL isopropanol each. The beads can be stored in iso- propanol at 4°C (away from light) (see Note 8). This section is illustrated in Fig. 2. 1. Remove 0.5 μL of a 10 mM bait compound stock solution and add 9.5 μL DMSO. This is the T0 time point. Add 10 μL MeOH to each sample (T0 and T16 ). 2. Inject 5 μL of the samples beginning with the T16 time point to avoid carryover. 3. The applied solvent system and LC conditions were: (a) Solvent A=0.1% formic acid in H2 O (b) Solvent B=80%MeOH/20% iProp (c) Flow rate=0.5 mL/min (d) 0–6 min 90% A/10% B to 100% B (linear gradient, curve 6) (e) 6–8 min 100% B (f) 8–8.5 min 100% B to 90% A and 10% B (linear gradient, curve 6) (g) 8.5–11 min 90% A and 10% B 3.2. LC-MS Analysis of the Coupling Reaction
31 3 Compound Immobilization and Drug-Affinity Chromatography 4. The column temp was 40°C. 5. The reaction was analyzed on a photodiode array detector (PDA) scanning a range of wavelengths between 210 and 500 nm. 6. The MS was set to scan a mass/charge ratio from 100 to 1,000, switching continuously between the positive and negative elec- trospray ionization (ESI) mode. 7. The MS parameters were: (a) Capillary voltage: 3.2 (ESI+/−) (b) Cone voltage: 32 (ESI+), 30 (ESI−) (c) Extractor voltage: 3 (ESI+), 4 (ESI−) (d) RF lens voltage: 0.3 (ESI+), 1.2 (ESI−) (e) Source temperature: 150 (ESI+/−) (f) Desolvation temperature: 400 (ESI+/−) (g) Desolvation gas (N2 ): 450 (ESI+/−) (h) Cone gas (N2 ): 50 (ESI+/−) (i) HM and LM resolution: 15 (ESI+/−) (j) Ion energy: 0.6 (ESI+) and 0.1 (ESI−) Fig. 2. LC-MS analysis of immobilization reaction of c-dasatinib. (a) UV chromatograms of T0 and T16 samples measured at 254 nm. The peak at a retention time of 3.00 min disappears after 16 h of incubation. (b) Positive mode electrospray MS spectra at 3.00 min retention time of the T0 and T16 samples confirming that the observed peak represents c-dasatinib, which completely disappears after incubation, i.e., couples to the activated matrix.
32 U. Rix et al. 1. Culture KU812 cells in 20 mL DMEM on a 15 cm dish. After cells approach confluence, centrifuge the cell suspension for 3 min with 300×g. Aspirate the medium and resuspend cells in 2 mL fresh medium. 2. Fill 20 mL DMEM in four new 15 cm dishes and add 0.5 mL of the cell suspension. After ca. 2 days cells approach conflu- ence and then need to be split again. 3. To harvest the cells, centrifuge the cell suspension, aspirate the medium, and wash with PBS. Shock freeze the samples in liq- uid nitrogen and store them at −80°C until further use or use them directly for lysis and the protein quantification. 1. Thaw pellet on ice and resuspend in appropriate amount of Lysis buffer (depending on pellet size) and pull it through a 0.9 mm syringe ten times (see Note 9). 2. Transfer the homogenate to a 15 mL Falcon tube and incubate on ice for 30 min. 3. Transfer lysate to an ultracentrifuge tube, balance tubes, and centrifuge for 10 min at 4°C with 20,000×g. 4. Transfer the supernatant to a fresh polycarbonate ultracentri- fuge tube, balance tubes, and centrifuge for 1 h at 4°C with 100,000×g. 5. Transfer supernatant to a fresh 15 mL Falcon tube and keep on ice. 6. Determine the protein concentration by the Bradford method using a freshly determined standard curve. Apply 0, 5, 10, 15, 20, 25, and 30 μL γ-globin and 0.5, 1, and 2 μL lysate to the cuvettes. Add 1 mL of the dye reagent (1×) to each cuvette, mix by careful vortexing, and measure the absor- bance at 595 nm. 7. Prepare 25 mg aliquots in microcentrifuge tubes. Use the lysates directly for the drug pulldown or shock freeze the sam- ples in liquid nitrogen and store them at −80°C until use. 1. Prepare the 1× Lysis buffer freshly and keep it on ice. 2. Dilute the thawed cell lysate (preferably, prepare cell lysate freshly) with 1× Lysis buffer to a total volume of 3 mL (for 25 mg total protein) transfer to Beckman ultracentrifuge tube, balance tubes, and centrifuge for 20 min at 4°C with 100,000×g. 3. Collect “total cell lysate” (TCL) sample (100 μg per Western blot), add the appropriate amount of 4× Laemmli sample buf- fer, and denature proteins at 100°C for 3–5 min. 4. Pipette 100 μL 50% drug-bead slurry (i.e., 50 μL settled bead volume=0.1 μmol drug) into a 2 mL Microcentrifuge tube 3.3. Cell Culture and Harvest 3.4. Preparation of Total Cell Lysate and Protein Quantification 3.5. Affinity Pulldown
33 3 Compound Immobilization and Drug-Affinity Chromatography (always use cut pipette tips for pipetting beads) and centrifuge beads for 3 min at 4°C with 75×g, remove supernatant. 5. Add 1 mL 1× Lysis buffer and wash beads by gently resuspend- ing and inverting several times, centrifuge beads, remove super- natant, repeat wash step three times, and remove supernatant. 6. Combine and resuspend the beads with the centrifuged cell lysate. 7. Incubate on roto-shaker for 2 h at 0–4°C with 10 rpm. 8. Centrifuge beads, remove and discard supernatant, but leave ca. 700 μL buffer in microcentrifuge tube (perform following steps in cold room at 0–4°C). 9. Resuspend beads gently and transfer to (plugged!) Mobicol columns, let beads settle by gravity. 10. Drain remaining buffer by gravity flow, fill with fresh 1× Lysis buffer and connect to 30 mL syringe with luer lock tip. 11. Add 7.5 mL 1× Lysis buffer and let the buffer drain by gravity flow (possibly apply gentle pressure with syringe plunger). 12. Place the column in a 2 mL microcentrifuge tube and centri- fuge for 1 min at 4°C with 100×g, plug the column, and place it in a 1.5 mL microcentrifuge tube. 13. Add 30 μL 4× Laemmli sample buffer and denature proteins at 100°C for 3–5 min. 14. Open first the top lid of the column, then unplug the bottom (careful!) and place back into the microcentrifuge tube. 15. Centrifuge for 1 min at room temperature with 400×g, collect eluate (this is the first elution sample) and replug the column. 16. Place column in another 1.5 mL microcentrifuge tube, add 30 μL 4× Laemmli sample buffer and denature proteins at 100°C for 3–5 min. 17. Unplug the column, centrifuge (as before) and collect eluate (this is the second elution sample) (see Note 10). 18. Store at −20°C until SDS-PAGE analysis. 1. For the 7% separating gel, mix 2.5 mL SDS buffer, 2.35 mL 30% acrylamide, 5.17 mL H2 O, 100 μL 10% APS, and 10 μL TEMED and fill ca. 4 mL (leave space for 2.5 mL of stacking gel) of the resulting solution between the two glass plates and overlay with 1 mL isopropanol, wait until the gel has polymer- ized (ca. 20 min) and pour off the isopropanol. 2. For the stacking gel, mix at first 25 mL Gel buffer 2 with 16.6 mL 30% acrylamide and 58.4 mL H2 O. Take 10 mL of this solution and add 100 μL 10% APS and 15 μL TEMED. Pour ca. 2.5 mL on the polymerized separating gel and insert a comb. 3.6. One-Dimensional SDS-PAGE
34 U. Rix et al. 3. After another 20 min remove the comb and place the gel in the electrophoresis chamber, which is filled with running buffer. 4. For immunoblotting, load 10 μL (5 μl each of first and second elution) per well, in the first well inject 5 μL of the protein marker. For silver staining, mix 10 μL of the first and second elutions and alkylate the sample with iodoacetamide (final con- centration 13 μg/μL) for 20 min in the dark. Then load the samples on the gel. If possible, fill the first and the last well of the gel with 8 μL of Laemmli sample buffer to ensure an even running profile. 5. Complete the assembly of the gel unit and connect the power supply. The gel can be run at 120 V or higher, if time is short. When the lysates are run through the gel it can be transferred to the nitrocellulose membrane or used for silver staining. This section is illustrated in Fig 3a. 1. Perform all the following steps in a dust-free cabinet to mini- mize keratin contamination. First, fix the gel with 40% ethanol and 10% acetic acid (always add sufficient solution to com- pletely cover the gel) for 1 h at room temperature on a rocker or at 4°C overnight. 2. Wash the gel two times with 30% ethanol for 20 min and for another 20 min with water (pro analysi). In the meantime prepare the sensitizer, the silver nitrate (refrigerator) and the developer. 3.7. Silver Staining Fig. 3. SDS-PAGE-based analysis of the dasatinib pulldown eluate from KU812 CML cells (9). (a) Silver staining shows several bands that subsequent LC-MS/MS analysis reveals to correlate with validated dasatinib target kinases. (b) Immunoblot with anti-ABL 21–63 antibody shows the enrichment of BCR-ABL and c-ABL by the dasatinib affinity matrix while an ampicillin control matrix does not display affinity for the dasatinib cognate targets. SFK SRC family kinase.
35 3 Compound Immobilization and Drug-Affinity Chromatography 3. Sensitize the gel with 0.02% Na2 S2 O3 for 1.5 min and wash it three times for 20 s with water (pro analysi). 4. Incubate the gel with refrigerated 0.2% AgNO3, for 20 min. 5. Discard the silver nitrate in heavy metal waste and wash the gel again (three times for 20 s). Transfer the gel into a clean Petri dish after the second wash step to keep the background as low as possible. 6. Develop the gel in 3% Na2 CO3 and 0.05% formaldehyde. Rock the Petri dish until the solution changes to a yellow color. Remove the liquid and allow the development without liquid. If the staining is not sufficient, repeat the developing step. 7. When all the bands are visible on the gel, quench the developer by removing the liquid from the Petri dish and adding 5% ace- tic acid. Leave the gel in the acetic acid for a minimum of 5 min. 8. Remove the acid, place the gel in the lid of the Petri dish, cover it with parafilm, and scan the developed gel for the documen- tation using a conventional picture-scanner. 9. Cover the gel with water if the gel is stored in the refrigerator for further analysis. This section is illustrated in Fig. 3b. 1. Take three 8×9 cm filter papers, soak them in 1× Western blot buffer (containing 10% methanol), and place them in the transfer unit. Then take a 7×8 cm membrane, soak it in the buffer, and place it on the filter paper. Cut away the comb of the gel, soak the gel in the buffer, carefully lay it on top of the membrane, and cover it with another three soaked filter papers. Finally ensure that there are no air bubbles in the resulting sandwich and close the transfer cassette. The transfer can be accom- plished at 56 mA (1 mA per cm²) for 1.5 h. 2. Incubate the membrane for 1 h in approximately 50 mL Blocking buffer. 3. Discard the Blocking buffer and incubate the membrane with the primary antibody dilution over night in the cold room. Use gentle agitation. 4. Wash the membrane three times for 5 min with PBS-T. 5. Add 10 mL of the secondary antibody dilution (always use a fresh solution) and incubate for 1 h. 6. Discard the solution and wash the membrane again three times for 5 min. 7. Mix together the ECL Plus solution (1:40) and cover the membrane with 2 mL of the resulting solution. Incubate for 4 min and let the redundant ECL solution drip down. Envelope the membrane with the Saran-Film (ensure that there are no 3.8. Immunoblot
36 U. Rix et al. bubbles) and place it in an X-ray film cassette. Perform the remaining steps in a dark room under safe light conditions. 8. Insert a developing film in the cassette and expose the film for a few seconds to minutes (see Note 11). 1. Always use cut pipette tips for pipetting beads with a clean scalpel or scissors. 2. Use high-purity distilled water of consistent quality to avoid contaminations, such as DNase and RNase free distilled water (Gibco). 3. The primary antibody and therefore the blocking conditions depends on any prior knowledge of validated target proteins of the bait compound. For our example, we have chosen dasat- inib as the bait compound, the cognate target of which is ABL. Therefore, we will delineate in the following conditions for the anti-ABL (21–63) antibody (Santa Cruz Biotechnology). 4. The primary antibody can be often reused for subsequent applications. If used in milk/PBS-T, it should be stored at 4°C and be used within a few days. If used in BSA, it can usually be stored at −20°C for several months. 5. Always use aerosol filter pipette tips to avoid cross contamina- tion of samples, as mass spectrometers are highly sensitive instru- ments, which will detect even trace amounts of contaminants. 6. Contamination of samples with keratin is one of the most com- mon and serious complications for proteomics mass spectrom- etry analysis, due to the abundance of this family of proteins and dynamic range limits of mass spectrometers. There are sev- eral sources of keratin contamination, such as dust, hair, skin, and clothing. It is therefore essential to take the precautions to reduce keratin levels as much as possible. (a) Always wear gloves and laboratory coat as much for per- sonal protection as for avoiding keratin contamination. (b) Avoid wearing woollen clothing, but rather synthetics or cotton. (c) Use a designated dust-free clean bench. (d) Use a dedicated set of pipetting and electrophoresis equipment. (e) Prepare buffers and reagents freshly from designated stock solutions. 7. The bait compound needs to contain either a primary or sec- ondary amino group, as does c-dasatinib, which was designed 4. Notes
37 3 Compound Immobilization and Drug-Affinity Chromatography based on the available X-ray co-crystal structure of dasatinib with ABL (10). Alternatively, primary or secondary alcohols can be readily esterified under Steglich conditions, i.e., in the presence of dimethylaminopyridine (DMAP) and dicyclohexy- lcarbodiimide (DCC) (5). For example, the hydroxyl group of dasatinib can be esterified with N-Boc-glycine. Subsequent removal of the Boc protecting group with trifluoroacetic acid yields a primary amine suitable for immobilization. 8. Duration of storage depends strongly on the stability of the bait compound. Although the dasatinib matrix was sufficiently stable, our experience with other compounds suggests that a storage of maximum 2 weeks should not be exceeded. If pos- sible, drug beads should be used immediately. 9. Some primary cells express high levels of protease activity which may require the addition of additional protease inhibi- tors to the Lysis buffer, such as the Complete Protease Inhibitor Cocktail Tablets (Roche). 10. Samples from the first and second elution are stored separately, but can be combined directly before loading on SDS-PAGE gel, if desired. 11. Exposure time typically varies. Acknowledgements This work was supported by the Austrian Federal Ministry for Science and Research (BMWF) under the GEN-AU program (GZ BMWF-70.081/0018-II/1a/2008) and the Austrian Academy of Sciences (ÖAW). References 1. Oda, Y., Owa, T., Sato, T., Boucher, B., Daniels, S., Yamanaka, H., Shinohara, Y., Yokoi, A., Kuromitsu, J., and Nagasu, T. (2003) Quantitative chemical proteomics for identifying candidate drug targets, Anal Chem 75, 2159–2165. 2. Rix, U., and Superti-Furga, G. (2009) Target profiling of small molecules by chemical pro- teomics, Nat Chem Biol 5, 616–624. 3. Han, S.-Y., and Kim, Y.-A. (2004) Recent development of peptide coupling reagents in organic synthesis, Tetrahedron 60, 2447–2467. 4. Anderson, G. W., Zimmerman, J. E., and Callahan, F. M. (1963) N-Hydroxysuccinimide esters in peptide synthesis, J. Am. Chem. Soc. 85, 3039. 5. Neises, B., and Steglich, W. (1978) Simple method for the esterification of carboxylic acids, Angew. Chem. Int. Ed. Engl. 17, 522–524. 6. Kocienski, P. J. (2005) Protecting Groups, 3rd ed., Thieme, Stuttgart. 7. Rix, U., Hantschel, O., Durnberger, G., Remsing Rix, L. L., Planyavsky, M., Fernbach, N. V., Kaupe, I., Bennett, K. L., Valent, P., Colinge, J., Kocher, T., and Superti-Furga, G. (2007) Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel kinase and nonkinase tar- gets, Blood 110, 4055–4063. 8. Lombardo, L. J., Lee, F. Y., Chen, P., Norris, D., Barrish, J. C., Behnia, K., Castaneda, S., Cornelius, L. A., Das, J., Doweyko, A. M.,
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wines, wool, and other products are numerous, but unimportant. The iron ore mines (red and brown hematite) in the Somorrostro range and district are largely in the hands of English capitalists. These mines, which began to attract the attention of British iron masters about 1870, occur chiefly in the mountain limestone, and are worked in open quarries. Short railways and tramways have been made to San Nicolas on the Nervion; and a wire tramway has been constructed by the Galdames Mining Company, who possess a cliff of iron ore about a mile long and 280 feet high. The tramway carries the ore through a tunnel, 600 feet long, to the quay. The Landore Siamese Steel Company have important hematite mines connected with the river by a wire tramway, carrying baskets for loading. BILBAO—THE ORCONERO IRON ORE COMPANY’S WHARF IN LUCHANA. Bilbao is largely modern and wholly commercial, and its public buildings are not notable. But its thoroughfares are full of movement, and the shady arenal, in the old town—the focus of the life of the whole city—contains the principal hotels, the chief cafes, and the New Theatre. The land which this beautiful promenade now occupies was at one time very boggy, and swept by the tides. Now the two principal avenues are asphalted. The Church of San Nicolás de Bari, which faces it, is one of the city parish churches. It was built towards the end of the fifteenth century on the ruins of the sailors’ and fishermen’s little church. This church has suffered greatly on account of floods, especially during the year 1553. It was closed in 1740 as ruin threatened it. When it fell, the present one was begun in 1743. During the last war it was used as a provisioning station; and, after repairs, was opened for worship on the 21st of January, 1881.
A GALICIAN. T In Northern Spain. HE great bulk of the Spanish people know as little of Galicia and the neighbouring Principality of the Asturias as the average Englishman knows of the Hebrides. Nor can they judge of the inhabitants of these provinces from the few individual Galicians who emigrate to Madrid any more than we in England can form an idea of Italians from the specimens who perambulate the London streets with a piano organ and a monkey. The Madrileño comes across a few Galicians in the capital engaged in menial services, and speaking a harsh, strange patois, which he finds some difficulty in understanding; but the Gallegan in exile is a very different person from the man you meet in his own land of rain and mist, where the scenery is exquisite, the hotels are famously bad, and devotion is the chief recreation of the community. At home these people are poor, but hardy; possessing little intelligence, but great capacity for work; knowing little comfort, but nursing a passionate attachment for the country of their birth. Many of the young women are remarkably handsome, but drudgery and hardship early tell their tale, and very few of them retain their good looks beyond the age of twenty. The country, for the most part, is poor to barrenness; the peasantry work day and night for mere subsistance; the cottages, which do duty for bedroom and nursery, stable, kitchen, rabbit hutch, pigsty and parlour, are damp and dirty, and destitute of beds or chimneys. The climate is rainy, the surface is mountainous, and the roads are generally bad. Small wonder is it that muleteers and commercial travellers constitute the principal visitors to Galicia—for those who have a soul above scenery, and an ambition beyond fishing, the country is practically without attraction. The single province of Oviedo, which constitutes the principality of the Asturias, harbours a people who have remained unconquered alike by Roman and Moor. There is protection, if not complete safety, in a country of mountain and valley, of damp and cold; and the Asturians have ever been able to spread themselves over the land and farm their straggling holdings in comparative security. They have cultivated maize for their staple food,
A GALICIAN. A GALICIAN. poached the hills and rivers for game and fish, cultivated the art of dancing, and lived in terror of the evil eye from the most ancient times; and despite damp, hard fare, and harder toil, they have learnt REDONDELA (PROVINCE OF PONTEVEDRA)—GENERAL VIEW. the secret of longevity and the charm of a gracious civility of manner. Minerals in abundance are common to both Asturias and Galicia; and while the former is the richer in coal and iron, the latter has been worked for gold, silver, and tin from the time of the Roman occupation. It is on their mineral resources that these provinces will have to depend for their future prosperity.
IN GALICIA. IN GALICIA. After the cities of the South— Barcelona, Toledo, Granada, or even modern Madrid—the Northern towns are small, shabby, and unimportant. Coruña, the chief seaport of Galicia, though interesting to Englishmen as being the landing place in Spain of John of Gaunt, and the harbour from which the invincible Armada sailed to conquer and Romanise Great Britain, is a place of only secondary importance. The city was founded by the Phœnicians; its name is probably derived from Columna, the Phœnician Pharos, or lighthouse; and its famous lighthouse, the Tower of Hercules, has had its counterpart from the earliest days. The Phœnicians, who made gain rather than discovery the aim of all their expeditions, were attracted to Galicia and to the province of Orense particularly by reason of its rich deposits of tin. Coruña in ancient days was the principal port of the North-west Coast, and the most westerly town in Europe. It is still the chief military station in Northern Spain, and ranks as a commercial city of the first importance. CORUÑA—GENERAL VIEW TAKEN FROM THE OLD TOWN. The hill-girt city of Santiago, though knowing nothing of commercial prestige, and having no part in the military system of the country, is to the traveller of far more interest than the capital of the province. For dead as it
now appears to be, with the hand of death on its crooked, branching streets, and its crazy, deformed squares, which echo the pilgrims’ footfalls to the deaf ears of the dead, it was at one time the most celebrated religious centre in Spain—the goal of fanatics from every corner of Europe, the Mecca of countless thousands of theologians, and the tomb of one of the personal companions of Christ. Although the ancient glory of Santiago has departed, although PONTEVEDRA—GENERAL VIEW. its broad-flagged pavements are no longer thronged by the feet of the devout, and it has been much shorn of its former civil and religious dignities, the city is still the See of an Archbishop with a cathedral, two allegiate churches, and fifteen parishes. The cathedral is erected on the site of the chapel which was erected by Alonso II. to mark the spot where Theodomer, Bishop of Iria Flavia, is said to have discovered the body of St. James the Apostle; and the city, which sprang up around the memorial, bears the Spanish name for St. James the Elder. The original cathedral, which was finished in 879, consecrated in 899, and destroyed by the Moors in 997, was replaced by the present edifice in 1078. Whether one believes or not the tradition of the foundation of the cathedral—which, by the way, is no mere
tradition in the mind of the Galician—one cannot but regard this mighty pile of stone with awe, and recognise in it the expression of an influence which was once felt throughout the Christian world. Even to-day it is one of the most frequented pilgrim-resorts in Europe. One passes through Pontevedra, a picturesque granite town, with arcaded streets and ancient houses bearing armorial shields, on the journey to Vigo. Here, as everywhere on the Galician coast line, the parish priest goes down to the shore one day in every year and blesses the sea; here also the oysters are excellent and abundant, and here the watchman’s night chant is heard in the streets. The call of the sereno, or watchman, who dates from the building of the ancient walls of Pontevedra, and the chapel of Alonso II. of Santiago, seems to catch the imagination of the traveller, and hurl him back into the mediæval ages, when life was a state that men fought to retain, and religion was a power for which they laid it down. The sereno, with his theatrical cloak wrapped about him, his axe-headed staff, his lantern, his majestic stalking walk, and his thrilling chant, “Ave Maria Purissima. Son las diez y sereno,” seemed to me impressive, unreal, almost fantastic. At ten o’clock he passed me in the deserted square, at eleven he was offering up his quavering invocation beneath my window. Galicia has little in common with the towns of the South—it retires to rest early in order to be up betimes. At Vigo a small fragment of the ancient sea walls yet remain, but the ruins that Lord Cobham made of the town in 1719 have been obliterated, and in place of the fortified port, which Drake visited in 1585 and 1589, we have a thriving, modernised town. Vigo is an important place of call for Mediterranean steamers, it is one of the chief centres of the cattle trade export to London, and the port of the mineral provinces of Pontevedra and Orense. The town of Orense, the capital of its province, is reached by the magnificent old bridge that spans the river Miño. Though now deprived of three of its arches, which were removed to give the road more width, and also of the ancient castle which defended the entrance, it continues to attract the attention of the traveller on account of its elegant and bold construction, its ample proportions and majestic appearance. Tradition says it is Roman, but many learned writers find nothing to confirm this assertion. It is quite likely that a bridge existed there previously; but the present one, it would appear, was built by order of Bishop Lorenzo during the first half of the thirteenth century, and has since undergone many alterations, including those
to the largest arch, which is more than forty-three metres in width, and the reconstruction of which was completed about the middle of the fifteenth century. In the Roman days Orense was celebrated for its warm baths. These three springs, which are still in existence, flow copiously from fountains one above another, but the waters have lost their medicinal virtues—it is VIGO—VIEW FROM THE CASTLE. only a supposition that they ever possessed any—and are now used for domestic purposes. The present cathedral, which is an obvious imitation of the cathedral at Santiago, was raised in 1220. The cathedral, the warm springs, and the bridge over the Miño, comprise the three marvels of the city.
GIJON—THE WHARF. Equally ancient, but in many ways more interesting, is the capital town of Lugo. It boasts a cathedral which shares with San Isidoro of León the immemorial right to have the consecrated Host always exposed; Roman walls in an excellent state of preservation that entirely surround the city, and an establishment of baths. The bath-house contains 200 beds; and the springs, which contain nitre and antimony, are good for cutaneous diseases and rheumatism. The river Miño, which is the glory not only of Lugo but of Galicia, rises in the mountains, some nineteen miles from the city. As the centre of a beautiful and variegated country, which affords good sport for the angler, and scenery of enchanting loveliness to attract the artist, Oriedo, the capital of the Astionas, has its charms; but the seaport of Gijon, with its tobacco manufactory, its railway workshops, its iron foundry, and glass and pottery works, is a much more thriving and important town. Gijon, like Santander, is a flourishing port; and both have gained immensely in importance of late years. While the latter, with its handsome modern houses, makes a more splendid show, its drainage and sanitary arrangements leave much to be desired, and the harbour at low water is sometimes most offensive. Both towns are of Roman origin, but Gijon is the most pleasantly situated on a projecting headland beneath the shelter of the hill of Santa Catalina, and the harbour is the safest on the North Coast. It exports apples and nuts in enormous quantities, coal, and iron, and jet; while its shores are much frequented by bathers during the summer months.
SANTANDER—THE PORT. SANTANDER—GENERAL VIEW. It is currently believed, and I have no reason to doubt the accuracy of the statement, that if a visitor in any town in England stops the first native he meets and inquires as to the objects of interest that the place possesses, he will be referred immediately to the principal hostelry of the town. If you wander in London, and ask your way about, you will be directed right across the city by references to public-houses, which are the only landmarks that the Cockney ever dreams of studying. In Spain, cathedrals are as ubiquitous as inns are in England. You may be sure of finding comfortable accommodation for man and beast in most English towns, and in the Peninsula you can be quite as confident of “bringing up” against a cathedral —if nothing else. In León, the capital of the province of the same name, and in Salamanca, the second city in the province, we find the same state of things existing—the cathedral first and the rest nowhere. Yet these two cities boast of a noble history of ancient splendour and old-time greatness, and with this—and their cathedrals—they appear to be content. León, in the time
LEÓN—THE CATHEDRAL. LEÓN—CLOISTER IN CATHEDRAL. of Augustus, was the headquarters of the legion that defended the plains from the Asturian marauders; and when the Romans withdrew, it continued as an independent city to withstand the continued attacks of the Goths until 586. The city yielded to the Moor, was rescued by Ordoño I., and retaken by the Arabs with every accompaniment of inhuman atrocity. Its defences were rebuilt by Alonso V. nearly 400 years later, its houses were repeopled, and it continued to be the capital of the Kings of León until the court was removed to Seville by Don Pedro. Its present miserable condition is a lamentable appendix to such a history. Its streets are mean, its shops are miserable, and its inns are worse. Nothing is left to it but its cathedral. This temple is truly an architectural wonder, combining the delicacy of the purest Gothic style with a solidity which has stood for centuries; the manner in which the problem of stability was solved is wonderful, the immense weights seeming to have no solid bases. The finest and most beautiful chiselled work is visible everywhere, and careful study is necessary in order to understand how the weight and strain of the arches were made to rest on their elegant buttresses. The origin of this magnificent temple is not quite clear, but many archæologists believe that it was founded in the time of King Ordoño II. It is of irregular form, but the cathedral or nave, transept, and presbytery are in the form of a perfect Latin cross.
LEÓN—THE CATHEDRAL CHOIR STALLS. The windows are of colossal dimensions, and the ratablos and sculptures are notable. Among its many famous works the cloister must not be forgotten. It is an example of the transition style from ogive to renaissance, with large galleries, interesting groups of sculpture, and a beautiful door leading into the temple. LEÓN—VIEW TAKEN FROM THE CEMETERY. Among all the choral stalls treasured in Spanish churches those in the cathedral at León stand out prominently. Unfortunately, the names of the master who designed them, and of the artists who assisted him to carry that marvel of ogive art into effect, are not known; but it must have been executed during the last thirty years of the fifteenth century, for it is known that in 1468 the necessary bulls were obtained from his holiness through Archbishop Antonio de Veneris in order to arrange means for meeting the cost of the stalls, and in 1481 the work was still proceeding.
SALAMANCA—GENERAL VIEW. Salamanca has a great name, a florid Gothic cathedral, and a square of handsome proportions and pleasant prospects. In other respects, it is quite without attractions. The streets are badly paved and dull, the climate is shrewd, and fuel, I was told, is scarce and expensive. Even the cathedral, though grand, is bare; and when one has visited the cathedral and lingered awhile in the pleasant garden of the Plaza Mayor—one of the largest and handsomest squares in Spain—and tested the accommodation of “La Comercio,” one can find little else to entrance one in the disappointing old city which was once a world-famed seat of learning. In the fifteenth century, when its university gave precedence to Oxford alone, it boasted of 10,000 students. In the following century its scholars had declined to one half that number, and to-day only some few hundred students are on its books. The sun of Salamanca commenced to set at a period of the world’s history that to all the rest of Europe was one of awakening and advancement. Decline and decay are writ large on the face of the city. From a distance its noble situation and fine buildings, built of beautiful creamy stone, gives the place an imposing and picturesque appearance. But though the shell of Salamanca remains, its spirit has departed. The ravages of the Romans, the Goths, the Moors, the Spaniards, and the ruin which the neighbourly French inflicted less than a hundred years ago, have left their cruel marks upon its historic walls. Salamanca is but a broken hulk spent by the storms that, from time to time, have devastated her. Her narrow, tortuous, ill-paved streets, which skirt its multitude of grandiose buildings, her squalor and poverty, her inferior art work, but even more the uncorrupted art of the grand old cathedral, all remind us of what Salamanca was, and turn our eyes backwards from what it is.
SALAMANCA—VIEW OF THE COLLEGE FROM THE IRLANDESES. ZARAGOZA—“INDEPENDENCIA” PROMENADE.
ZARAGOZA—PILAR CHURCH. One must approach Zaragoza with one’s mind full of memories of heroes, queens, poets, and bandits that have been associated with this once mighty city, and one’s heart filled with sympathy and respect for the old, proud Aragon that flourished, and was illustrious in history while the Englanders still decorated themselves with blue paint, and were domiciled in caves. For Zaragoza is not altogether a gay or an exhilarating city. Many of the streets have a gloomy aspect, and the old houses are high, dark, and repellant. But the city is not only important as the seat of a university, an Audiencia, an archbishop, the captain-general of Aragón, and other officials; it is also the junction of four railways, and its commercial progress has been steadily increasing of recent years. For Zaragoza is in reality two cities—the old part with ancient fortified houses, converted now into stables and wood stores, and the new part traversed by broad, well-paved, and excellently-lighted streets, and lined with modern buildings. Until the railway connected the city with Madrid and Barcelona, Zaragoza was as dead as Salamanca, and as dilapidated as León. But it has always held the advantage of those places in having two cathedrals to their one. The principal cathedral, that of La Seo, is a venerable Gothic pile occupying the site of a Moorish mosque, and its high arches have echoed many councils, and looked down on the solemn coronations of the kings of Aragon. More modern is the Cathedral El Pilar, so called from the identical pillar on which the Virgin descended from heaven. It was commenced on St. James’s Day, 1686, the work being designed and carried out by the famous Don Francisco Herrera, the architect. In the year 1753 King Ferdinand VI. instructed Ventura Rodriguex, the architect, to design and build a new church, as luxurious as possible, in which to instal the image without taking it out of its temple. This was done by erecting a small Corinthian temple under the magnificent cupola, which was ornamented with the richest marble and jasper that could be procured. On one of the altars of this temple, which is crowned with a magnificent silver canopy, reposes the venerated effigy, the jewels on which are of incalculable value.
A FLEMISH DANCE. AT NUEVALOS. The Stone Monastery at Nuevalos, on the right bank of the river from which it takes its name, is one of the places most worthy of a visit in the province of Zaragoza, not only on account of the building itself, which is of great historical interest, having been built in 1195, but for the delicious picturesqueness of the place. Surrounded by rocks, winding amidst thick woods and dashing into deep abysses, this river runs its erratic course, imparting life to a landscape which is, according to the noted poet, Don Ramon Campoamor, “an improved dream of Virgil.” Among its many picturesque waterfalls, the one called “La Caprichosa” is perhaps the most beautiful. The dress of the Aragonese peasantry is peculiar and picturesque. The men, as a rule, wear no hats, but have instead a coloured handkerchief wound round the head, leaving the top bare. Their knee-breeches are slashed down the sides and tied by strings below the knee. The waistcoats are worn open. Round the waist they wind a wide sash, in the folds of which pipes, tobacco, money, and provisions are carried as safely as in a pocket. Their feet are shod with sandals, and they universally carry a blanket, which is thrown in a graceful manner over their shoulders.
A Bull-fighting. BULL-FIGHT is underlined for an early visit in the note-book of every visitor to Spain. He goes prepared to be disgusted, and he comes away to denounce it as a revolting and demoralising exhibition. He even plumes himself upon his moral and human superiority over the Spaniard, because the spectacle proves too strong for his untutored stomach. The inference is as gratuitous as it is illogical. In point of fact, the effect of the spectacle upon the spectator is not so much a matter of sensibility as custom. The Spaniard grows up to the sport as our Elizabethan ancestors grew to bull-baiting—even as the present generation of Englishman grows to pugilism. To the Spaniard, the cruelty of the craft of tauromachy does not appeal; the spectacle inflames his blood, and stirs not a chord of compassion in his nature. Yet he can be intensely sympathetic, gentle, and tender- hearted; but these softer qualities of character are not touched by the sight of animal suffering. In the first place, the bull is his enemy by heredited tendency. He cannot forbear to hurl insulting epithets at him when he chances to pass him on a journey. He witnesses his end with the thrill of satisfaction which a soldier feels in the death of a treacherous and implacable foe. The Englishman cannot share, or even realise this sentiment —it would be strange if he could. His leading feeling is curiosity, and a nervous apprehensive tension which only magnifies the horror and repulsion of the sport. With the Spaniard it is entirely different. Long habit has familiarised him with the bloody details, and his experienced eyes follow each trick and turn of the contest with the enthusiasm of an athlete watching an athletic display. Every detail of skill and dexterity and nerve exhibited by the fighters, and every clever move made by the bull is greeted with critical applause. Cruelty there must be, but courage in a high degree is a factor in the contest—danger gives to the contest a dignity which is absent from pheasant shooting, and which formed no excuse for the vogue to which bear- baiting and cock-fighting once attained in this country.
THE PROCESSION. It may be thought that I am trying to champion an institution which is regarded with aversion by all classes of English people, but such is not my intention. My object is to look at it from the Spanish point of view, and endeavour to see if there is not some plausible explanation of its popularity as a national amusement. But when all is said and done, there still exist two objections to the sport which cannot be explained away. The first is the almost inexplicable indifference which a Spanish audience shows for the torture that is inflicted upon the horses that take part in the corrida: the other is the attendance of the gentler sex. It must, however, be noted that a large proportion—certainly the majority of Spanish ladies—are opposed to the sport, and with the rest it is the manly courage and address of the performers that fascinates them. But the fact remains that women are seen in large numbers in the amphitheatre, as 300 years ago good Queen Bess was not ashamed to be a spectator at many an exhibition of bear-baiting. English sentiments in matters of sport have undergone a great change since the Elizabethan era, but Spain is notoriously the most conservative country in Europe. However, enough has been said of the theoretical side of bull-fighting; let us accompany the seething populace to the Plaza de Toros, and witness the sport for ourselves. The streets of Madrid are crowded with people who are all moving in the same direction. April to October is the regular bull-fighting season, but the Spaniard finds the lightest excuse a sufficient one for indulgence in his favourite pastime during the “close” season. And so, although it is February when I am in Madrid, I am not to forego an experience of a promising corrida.
Although I have seen bull-fights in some of the best rings in Spain, including those of San Sebastian, Valencia, Barcelona, and Madrid, it is more particularly of my experiences at the latter place that I shall write. During the fashionable months, a boletin de Sombra, or “ticket for the shade,” is a luxury to be prized; but in February, in Madrid, we need all the warmth and glare that the sun can give us. The present Bull Ring, which was built at a cost of £80,000, and opened in 1874, seats 15,000 persons. It stands on a gentle elevation in a broad stretch of bare yellow land, where it raises its brick-coloured walls—the only land-mark in the barren, treeless, desolate expanse between the city and the solemn distant mountains. Around the various entrances countless human beings cluster like bees, and the Plaza is alive with men and horses, mules with tinkling bells, soldiers, police, picadors, and fruit-sellers. What strikes one most curiously about this concourse of human beings, both outside the bull-ring and within the huge amphitheatre, which rises tier above tier from the brown sand till it is almost lost in the vast expanse of blue above, is its single-mindedness, its patience, and the entire absence of horseplay. To a Spaniard this is not curious, but to the English spectator some familiar characteristic of a crowd appears to be absent. ENTRANCE OF THE BULL. Punctuality is not a strong trait in the Spanish character, but punctuality will be observed to-day. At the hour and the minute appointed, the President enters his palco, the signal is given, and the proceedings commence. The procession, headed by two caballeros, habited in black velvet, moves slowly across the ring to the front of the President’s seat. The two espadas in yellow and violet, and gold and green costumes respectively, follow the caballeros. After them come half-a-dozen stoutly-protected picadores, then eight
banderilleros, gay with a profusion of silk sashes, short breeches, and variously-coloured hose, and the rear is brought up by a posse of attendants, leading the mules, all bedecked in plumes and rich trappings, which are to drag off the carcases from the arena. The entrance of the glittering cavalcade is announced by a trumpet sound, and the President tosses the key of the toril into the ring. To the “new chum,” all this preliminary detail, commonplace and “circusy” as it is, is sufficient to strain the nerves, and expectancy changes to apprehension. The creak emitted by the opening of the heavy door of the toril intensifies the feeling. The clutch of curiosity with which the entire concourse awaits the entrance of the first bull is contagious. Instinctively one strains forward and catches one’s breath. Toro does not keep us long in suspense. There is a momentary lull, and then the bull dashes from his dark cell into the glint of the Spring sunshine. The novelty of the environment staggers him for a moment. He hesitates in the centre of the ring, and looks wildly around him. The arena is empty, with the exception of three picadores, who sit rigidly in a row on their sorry hacks, waiting for the bull to recognise their presence. Our first victim is a doughty warrior. He is as ignorant as the blindfold knackers—that would be dear at a pound a leg—of the fate in store for him. He may make a brave fight, kill horses, upset men, and leap the barriers with a heroic rush, but in twenty minutes his corpse will be coupled up to the mules, and fresh sand will be strewn on the red trail that will mark his last passage across the arena. The inevitableness of the outcome of the encounter, so far as the principal actor is concerned, is the least pleasing feature of the sport. The fox and the stag are
ANTONI O FUENTE S. LUIS MAZZANTINI AND CUADRILLA. U E R R I T A . B a n d i l l e r o . given a gambling chance, the grouse is not without hope, and the gladiator of the cock-pit may live to fight another day, but the bull is a doomed animal. Happily he is not capable of calculating the uselessness of his efforts. The horses stand but little better chance, and the picadores, despite their iron and leather greaves and spears, are paid to take risks. The art of the picador is displayed in the skill with which he avoids the charge of the bull, and turns him on to the next picador, who, in turn, will pass him on to the third. In this instance the manœuvre does not come off. The bull’s rush is met by the first picador with the point, but the horse he strides is too ancient to obey with sufficient celerity the rider’s injunction to swerve, and horse and man are rolled over with the force of the impact. The
wretched equine is lacerated on his opposing flank, but the spearman appears to be uninjured, and before the bull has completed his circuit of the ring, the horse is on his feet again, and the picador is waiting for the next attack. The toreros, with their red capa, are immediately on the spot to draw the bull from his victim, but the bull is too eager to waste time on a fallen foe. The second and third horseman avoid his rush; and the bull, smarting from spear thrusts, and confused by the cheers, is inclined, in racing parlance, to “turn it up.” The first horse who crosses the line of sight is caught on the brute’s horns, and is so deeply impaled that the bull has to swerve at right angles to rid himself of his enemy. The second horse is impaled before the combatant can plant his spear in the bull’s neck. Steed and rider are lurched in the air, and fall heavily to the ground, and the momentary victor lowers his head again to the prostrate man, and rolls him over and over. Toreros hasten to the spot to get him away, the people rise in their places, ladies lift their fans and avert their faces, while the air is filled with the usual murmur of lamentation which accompanies an accident. Both the other picadores are unhorsed before the President gives the signal for them to retire. Act one of this most realistic of sporting melodramas is over. The banderilleros now come forward. They are costumed like Figaro, in the opera of “Il Barbiere de Sevilla,” and their hair is tied into a knot behind. To the English spectator, this part of the performance is the most fascinating and least abhorrent of the entire piece. The banderillero inflicts no more pain on the bull than the humane angler deals out to the wily trout, and the agility and daring with which he addresses himself to his task is superb. His aim is to plant small barbed darts, or banderillas, on each side of the neck of the bull. The chulos, or apprentices, here open the ball by tantalising the animal, and working him up to a proper pitch of fury. Then the banderilleros circle round him, and one, standing full in his line of flight, “defies” him with the arms raised high over his head. If the bull stops, as he is doing now, the man walks composedly towards him. Then the bull lowers his head and makes his rush, and the athlete, swerving nimbly to one side, pins in his banderillas simultaneously. Again and again the maddened animal, frantic more from impotence than pain, makes his rushes from one tormentor to another. At each rush he receives further instalments of his hated decorations. Then one man bungles. He loses his nerve, or, failing to time the animal’s charge, shirks the onslaught. A howl of execration greets the exhibition, and the unfortunate baiter is tempted to more rash efforts. He seats himself in a
chair, and waits with suicidal calmness the rush of the bull. Just as the animal’s horns are thrust beneath him he jumps lightly up, manipulating his darts with miraculous precision, while the chair is tossed high in the air. Thunders of applause greet this venturesome feat, and the other banderilleros, warmed to their work by the plaudits of the public, vie with one another in deeds of coolness and “derring do.” One waits, alert but motionless, for the attacks of the charging bull, and as the galloping brute lowers his head to toss him, places his foot between the terrible horns, and is lifted clear over his onrushing enemy. Another, seizing hold of the lashing tail, swings himself along the bull’s side, and plants himself for one thrilling moment right between the horns. THE PICADOR. I once saw a banderillero, in response to the jeers of the crowd, take the darts, which are about two feet long, break them across his knee, and plant the stumpy weapons, with unerring precision, on each side of the neck of the bull. These feats appear to be fraught with infinite danger, and the agility with which the performers acquit themselves cannot be witnessed without a tremour of amazement and admiration. Several times the venturesome chulos escape death as by a miracle: they sometimes seem so close to their end when they vault over the barriers to avoid the pursuing bull, that they appear to be helped over the fence by the bull’s horns. One bull exhibits at this stage of the proceedings an emphatic disinclination to continue the fight. He paws the ground when the darts are driven home, but makes no show of retaliation, and the hoots and opprobrious epithets that are hurled at him by the populace fail to inspire him to renewed efforts. Then the banderillas de
fuego are called for. These are arrows, provided with fire crackers, which explode the moment they are affixed in the neck. In a moment the spectacle, which had worked me up to a high pitch of excitement, becomes intensely distasteful. The tortured animal, driven mad with fright and pain, bounds across the ring in a series of leaps like a kid. The people scream with delight, and I mentally wonder what kind of “steadier” the Spaniard resorts to when his stomachic nerve is affected by a detail of the exhibition. The firework display had not lasted long when the last trumpet sounded, and the espada walks forward to a storm of rapturous applause. The finale of the spectacle is approaching. The executioner comes alone: the bull, who has hitherto been tormented by a crowd of enemies, is now able to concentrate his whole attention on one object. Toro has become exhausted with his previous exertions, and he moves without his old dash. The espada studies his foe carefully, to judge the temper of the animal with which he has to deal. With his left hand he waves the muleta—the red cloak —to lure the beast into a few characteristic rushes and disclose his disposition. If he is a dull, heavy bull, he will be despatched with the beautiful half-volley; but if he proves himself a sly, dangerous customer, that is cunning enough to run at the man, instead of at the muleta, a less picturesque, but safer thrust must be employed. But our bull is neither sly nor leaden. He has recovered from his fright, and is quick to seize his opportunity to make a final effort before the stinging banderilleros return to distract him. Once or twice he thrusts his horns into the unresisting cloak, then gathers himself together for a final rush. The swordsman raises the point of his glimmering Toledo blade; while every nerve of his sinuous, graceful body quivers with the absolute constraint and concentrated effort that hold him. The duellists are both of the same mind. The espada has summed up his antagonist—he is levantados, the bold bull, a fit subject for la suerte de frente. The bull’s next rush is his last. The fencer receives the charge on his sword, which enters just between the left shoulder and the blade. The bull staggers, lurches heavily on to his knees, and rolls over, at the feet of his conqueror, vomiting blood. The assembled multitude rend the air with their cheers, the men yell applause, and every face is distorted with excitement and enthusiasm. The only indifferent person in the building is the espada. With a graceful and unassertive turn of his wrist, he waves the sword over his fallen foe, wipes the hot blood from the blade, and turning on his heel, approaches the
President’s box, and bows with admirable sang-froid. The team of jingling mules enter, and the dead bull is carried off at a rapid gallop. The espada walks composedly away, without another glance at the result of his handiwork. The superb imperturbability of these espadas always fills me with admiration. They accept the plaudits of the spectators with the same unconcern with which they hear the execrations that fill the air if they do not at the first attempt inflict the coup de grace. During the first corrida I attended, an espada failed to aim at the precise spot, and the bull tore up the sand in agony. The populace insulted the swordsman with jeers and howlings, but he remained perfectly cool and collected, and nerved himself with as much composure to his second and successful thrust as if he had been practising with a sack of potatoes in an empty arena. When I had been witness to the death of two bulls, I remarked to my Spanish friend that I had seen as much as I desired, and was quite ready to quit the spot. But my companion was a friend of long standing: he could be firm without seeming discourteous. “No! no!” he said, “you kept me in the theatre last night until ‘Don Juan’ was played to the bitter end: you shall remain to-day to reward me for my exemplary patience and respect for your wishes.” I saw five other bulls done to death during the afternoon. AT CLOSE QUARTERS. Although not to be compared with an ordinary corrida as a display of skill, and capacity, and artistic finish, a Royal bull-fight, such as Madrid saw on the occasion of the coronation of King Alfonso XIII., is more interesting as being a revival of the sport as it was originally practised. Bull-fighting to- day is a purely professional business, but in the knightly days of ancient Spain it was employed as a means to teach the chivalrous youth the use of
arms. In those days, mounted caballeros encountered the bulls in the ring with lances alone—a more dangerous pastime than is bull-fighting in its modern sufficiently hazardous form. Then the combatants were mounted on good horses, and their business was to save them and turn the bull, to kill the bull if possible, but, at the risk of their own lives, to protect their steeds from injury. It is recorded that in one Fiesta de Toros at the beginning of the sixteenth century, no less than ten young knights lost their lives. The corrida, Real con Caballeros en plaza—a Royal bull-fight with gentlemen in the arena—on the olden lines, that was held on May 21st, 1902, in Madrid, was fought by young officers and scions of noble families, who were attired in the gorgeous costumes of Spanish knights of the reign of Philip IV., and attended by their pages and grooms wearing the dress of the same period, and displaying the colours of the noble house which they served. On that occasion, the Paseo de las Cuadrillas, or preliminary procession of the bull- fighters across the arena to the strains of military music, was a most imposing sight. The Padrinos, the grandees who acted as supporters or godfathers of the knights, accompanied the fighters, followed by their mediævally-clad retinues, to the foot of the Royal box, and presented them to the King. The spectacle was strikingly brilliant, but the display was not to be compared with a professional bout. The horses of the cavaliers had evidently not been sufficiently trained for their work, and the best riding in the world could not bring them off scathless. Let me condense an account of the scene to convey an impression of what the present-day bull-fight has been derived from. When the procession had withdrawn, leaving only the chulos and the gallant caballeros in the arena, the door of the toril swung on its heavy hinges, and a splendid specimen of a bull, dungeoned for several hours previously in utter darkness, darted into the light of day, tearing up the ground with its hoofs, and ploughing the air with its horns. Suddenly, a horseman and his prancing steed vaulted into the centre of the ring—the charger, with flowing mane, full-veined ears and shapely head slanted forward—to meet the onrush of the goaded bull. The second picador seeing the bull worried and dazed by the tantalising assistants, scudded past on a swift, white racer, sitting gracefully in his saddle, and then turning deftly as he passed the great brute, plunged his lance into his neck, and whirled aside to avoid possible pursuit. But by sheer accident, the bleeding steer dashed
off in the same direction, caught the horse in the hindquarters, raising it on its forelegs and endangering the equilibrium of the rider. Before the scampering bull had time to recover from the compact, the second caballero, dashing up, had planted his lance deep into its neck. The white horse, stung with pain, made a wild rush, but was brought to hand by splendid horsemanship, and his rider urged him along, to inflict another wound in the animal’s head. Then two toreros advanced, beguiling and wearying the bull. By the time the bull had received the fifth lance in his neck, and the white steed had been twice wounded, the edge was taken off the keen thirst for violent emotions, and another torero unfolded his red capa, waved it to and fro until the bull swooped down upon him, and a moment later he was sprawling in the sand seemingly gored by the infuriated animal. The next minute the wounded steer tottered, dropped on its forelegs, and turned over on the sand, and a knife put a speedy end to its sufferings. The second bull, a black massive creature, appeared listless and faint, and made little effort to defend itself. It made one successful attack on the white charger; and, then, at the signal from the King, an amateur espada stepped forward. The attempt was a miserable failure. The young swordsman dedicated, in a few well-chosen words, the death of the bull to his sovereign, and after a dozen passes with the red capa, plunged the gleaming blade of Toledo steel into the animal’s neck, but so ineffectually that a storm of hisses resounded through the ring. The second attempt was still more awkward, the sword entering but a few inches. The sword was pulled out, and another effort, made amid groans and hisses, proved equally unsuccessful. A TURN WITH HIS BACK TO THE BULL. Although the madness had died out of the expiring brute’s eyes, and his forelegs were bending under him, the inexperienced torero seemed unable to
put him out of pain. However, he grasped the short, sharp knife, and unsteadily taking aim, plunged it into the neck. Another failure. Yells, groans, shrieks, whistling, and hissing marked the anger of the crowd. The espada may be a paid professional, or the greatest noble in Spain, but in the ring he is judged by the rules of the ring, and his bungling is recognised with the most poignant scorn to which failure could be subjected. He again grasped the sword; and, spurred by the vitriolic exclamations of the public, sheathed it in the bull’s neck. The animal stood still and tottered, his forelegs bent, his head sank upon the moist, red sand, his hind feet quivered, and a flourish of trumpets announced that life was extinct. It is curious to find, in talking with learned enthusiasts on the relative merits of the bull-fighters, what diversity of opinion exists; but all parties are agreed upon the unrivalled skill and daring of the mighty Frascuelo. In his day, for death’s whistle summoned him from the arena in the height of his fame, Frascuelo was regarded as the greatest matador that Spain had ever seen; and Spaniards, in debating the subject of the bull-ring, never indulge the hope that his equal will ever arise to shed a new glory on the National sport. Frascuelo is dead, and his famous rival, Guerra, or Guerrita—to give him his professional name—has long since cut off his coleta, and lives in well-earned retirement at Córdova. But the school of fighters, who claim Frascuelo as their master—the fearless, dare-devil toreros, who scorn to concede a yard of ground to the bull, and do all their fighting at close quarters—is widely popular; and if their terribly dangerous methods are attended by frequent casualties, the intoxicating applause that rewards the accomplishment of a brilliant coup is, apparently, ample compensation for the risks that it entails. But the wildest appreciation of a successful feat does not exempt the most popular performer from the furious condemnation of the multitude when his scheme miscarries. The allowances made by a Spanish audience at the ring-side are of the most grudging nature. I once travelled from Barcelona to Madrid in the company of Bombita-Chico—the boy Bombita—who, although he was barely recovered from an unfortunate encounter with a tricky bull eight days before, was on his way to take part in a grand corrida that was to be held in the capital. He was—as his name denotes—no more than a lad, with large, strong hands that sparkled with jewels, while a formidable anchor about five inches long, set with magnificent diamonds, dangled from his watch-chain. I saw him again in the arena a few days later. He seemed nervous, and was, it appeared to me, a
little perturbed by the demonstration that welcomed his reappearance in the ring after his accident. Ill fortune allotted him a troublesome animal, and his kill, while creditable enough to untutored eyes, lacked the grace and finish that the critical spectator requires. Bombita was their own Boy of Madrid, and because of his recent misfortune they forgave him, but they did not cheer him; and the lad walked out of the arena amid a silence that could be felt. FIXING THE BANDERILLAS. Mazantini, now grown old and heavy, was in his day an undoubtedly fine matador. There are some that still regard him as the head of his profession. But the majority, remembering what he was, regret that he has not gone into honourable retirement. But Mazantini cannot tear himself away from the fascination of the arena, although his appearances grow less frequent every year. Conejito, who was wounded in Barcelona in the spring of 1903, is generally regarded as the most accomplished matador now before the public; but Fuentes is, par excellence, the best all-round man. For, with the exception of the picador business, Fuentes plays every part in the piece. Other espadas have their assistants, who play the bull with their capas, and stand by while the banderilleros ply their infuriating darts. It is only when the bull has been prepared for the slaughter by the other performers that the matador comes forward to put the finishing touch to the grim tragedy. Fuentes, on the other hand, on special occasions—of which the corrida which I attended in Madrid was one—keeps his assistants entirely in the background; he takes the stage when the picadores leave it, and keeps it to the end. So close does he keep to the bull, that during the corrida in Madrid, of which I am writing, he seldom allowed the animal to be a dart’s length away from him. On one occasion his capa got caught so tightly on the bull’s
horns that he tore it in jerking it away; and at another time the bull stopped dead, with his forefeet on the hated sash. As a banderillero, Fuentes is without equal in Spain. He frequently works with darts that have previously been broken short, and he uses them sparingly. Yet the encounter between the banderillero and the bull when Fuentes is on the scene is the most thrilling part of the whole performance. It is a contest between human intellect and brute intelligence—a duel between mind and matter. Fuentes does not avoid the bull, but by exerting some magnetic power he repulses the animal and compels it to halt. When the bull charges, in response to his “defiance,” he waits with the banderillas suspended above his head until the animal is within a few yards of him. Then he deliberately, but without haste, lowers one arm until the arrow is on a level with the brute’s eyes. The bull wavers in his onslaught, slows up, and stops dead within a foot or two of the point. Sometimes Fuentes walks backwards, while the bull glares at him with stupefied impotence, until he escapes the eyes that THE MATADOR. hold him, and gallops away. Again and again the banderillero taunts his enemy to attack him, only to arrest his charge and force him to turn from his deadly purpose by the irresistible power of his superior mentality. The crowd follows this superb exhibition with breathless interest, and in a silence that is more eloquent of admiration than the wildest cheers would be. But the end is nearly reached. Fuentes grasps his stumpy darts and advances against his bewildered antagonist, who waits his approach with sulky indifference. The man’s arms are flung up with a gesture of exasperating defiance, and when the bull makes his final rush, his opponent, instead of stopping him, steps lithely on one side, and the brute thunders past him with the two galling arrows firmly implanted in his huge neck. Fuentes has already moved to the
side of the ring. The bull turns and charges back at him. The banderillero glides gracefully over the sand, but his pace is not equal to that of his infuriated pursuer. The distance between them decreases rapidly; in half-a- dozen yards he will be upon him. Fuentes glances over his shoulder and, without changing his pace, doffs his cap and flings it in the bull’s face. This stratagem only arrests the rush of the brute for a moment, but it gives the man time to reach the barrier, where he receives his muleta and sword from an attendant and returns to complete his task. All the kings of the bull-ring have their own particular feats or strokes, which the Spaniards appreciate as Englishmen revel in Ranjitsinhji’s acrobatic hitting, or Morny Cannon’s inimitable “finishes.” Bombita-Chico’s speciality in playing his bull is to kneel in the arena and allow the animal to charge through the capa which is held within three feet of the ground. The nerve required for this feat fires the audience with enthusiastic approval. The tale is told of a torero, whose name I have forgotten, who gained distinction by his exceptional skill in facing the bull with the long vaulting pole, known as the salto de la garrocha. With this instrument he would goad the bull on to the attack. When the brute was in full gallop he would, timing his movements to the instant, run a few yards to meet him, and swing himself high into the air at the end of his pole. The oncoming bull would charge the pole, the grounded end would be tossed upwards, and the torero would drop lightly to the ground and make good his escape. On one occasion the man performed his risky “turn” at a moment when the attention of a royal lady was attracted from the arena, and she sent an attendant to the expert to command him to repeat it. In vain the poor fellow protested that it was impossible for him to accomplish the same feat again with the same bull. The lady’s desire had been expressed. “But it is more than my life is worth,” argued the athlete. “It is the lady’s wish,” responded the attendant. The torero bowed, and “I dedicate my life to Her Royal Highness,” he said. The attempt fell out as he foretold. The bull charged and stopped dead. The man vaulted aloft, his body described a half circle, and fell—on the horns of the bull. He was dead before the attendants could entice the animal from his victim.
THE FINAL STROKE. Lagartijo, Lagartijillo, Mazantini, and Montes all have their distinguishing methods of attacking and despatching the bull, but none of these are capable of the feat by which Guerrita was wont to throw the bull- ring into transports of deafening enthusiasm. In the ordinary way, the espada having taken the measure of his adversary, receives him standing sideways, and having thrust his sword at arm’s length between the left shoulder and the blade, leaps aside as the bull blunders forward on to his knees and falls to the earth. But Guerrita advanced his left arm across his body and waved his muleta under his right uplifted arm. When the bull lowered his head at the charge he passed the sword over the animal’s horns and plunged the blade into the vital spot behind the shoulder. In other words, he stopped the brute and killed him while his head was under his arm; and so closely were the duellists locked in that last embrace, that Guerrita’s side was frequently scratched by the bull’s horns. One may lecture, write, and preach against the barbarity of bull-fighting; but so long as Spain can breed men of such amazing nerve, and skill, and dexterity that they can successfully defy death and mutilation to provide their countrymen with such lurid sport, so long will bull-fighting continue to flourish in Spain.
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  • 6. Me t h o d s i n Mo l e c u l a r Bi o l o g y ™ Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651
  • 8. Chemical Proteomics Methods and Protocols Edited by Gerard Drewes and Marcus Bantscheff CellzomeAG,Heidelberg,Germany
  • 9. Editors Dr. habil. Gerard Drewes Cellzome AG Heidelberg, Germany gerard.drewes@cellzome.com Dr. Marcus Bantscheff Cellzome AG Heidelberg, Germany marcus.bantscheff@cellzome.com ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-363-9 e-ISBN 978-1-61779-364-6 DOI 10.1007 /978-1-61779-364-6 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011937567 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or ­ dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)
  • 10. v Preface An Overview of Chemical Proteomics: Methods and Applications The multidisciplinary science of chemical proteomics studies how small molecules of synthetic or natural origin bind to proteins and modulate their function. Scientists in the field have different backgrounds including molecular and cell biology, biochemistry, phar- macology, organic chemistry, and physics. Chemical Proteomics: Methods and Applications is directed at molecular biologists and biochemists with either an interest in small molecules themselves, e.g., in drug discovery projects, or in using small-molecule probes as research tools to study protein function. The book may also be useful for organic chemists with an interest in biology and for specialists in protein mass spectrometry. In the introductory chapters, we discuss analytical strategies for chemical proteomics projects, with a focus on the current state-of-the-art in protein mass spectrometry, and describe several examples how chemical proteomics can impact the field of drug discovery. The consecutive chapters provide detailed experimental protocols. Most chemical proteom- ics projects consist of three parts. In the first part, the chemical probe is selected or designed, and then synthesized. In the second part, the probe compound is exposed to the protein sample or cell extract. In the final part, proteins binding to the probe compound are identi- fied and often quantified. In simple applications, this is often achieved by antibody-based detection, but if the aim is the discovery of targets in an unbiased fashion, mass spectrom- etry is the method of choice. The following chapters cover all of these aspects. The first set of chapters describes how probes are generated from commercially available reagents without elaborate chemical synthesis procedures, and how the proteins binding to the probes can be analyzed by immunodetection or by mass spectrometry. Rix et al. and Saxena provide protocols for direct noncovalent affinity capture using protein kinase inhibi- tors as an example, which serves to profile the targets of these compounds and provides probes for kinase expression and activity. Ge and Sem have developed a target class-specific probe for the labeling of dehydrogenase enzymes. Kawamura and Mihai, and Codreanu et al. describe the use of biotin-conjugated probes which form covalent adducts with defined subproteomes, here consisting of adenine-binding proteins and targets of lipid electrophiles. Lenz et al. combine features of noncovalent and covalent capturing strategies in their bifunc- tional ligands designed to target methyltransferases, an emerging class of drug targets. The second set of chapters is concerned with techniques for the discovery of small- molecule targets and the probing of target function. Ong et al. describe the use of stable isotope labeling of amino acids in cell culture (SILAC) in identifying proteins that bind small-molecule probes in cell extracts. Hopf et al. perform affinity enrichment of target pro- teins on a probe matrix in the presence of competing free test compound in solution, thus enabling determination of binding potencies of the free test compound to affinity-captured proteins from cell extracts. The method employs quantitative mass spectrometry with iso- baric mass tags to determine the potencies for a large number of targets in a single analysis. Ge and Sem have developed a protocol for the detection and purification of dehydrogenase
  • 11. vi Preface enzymes, which may represent targets, but also unwanted off-targets, for certain types of drugs. Kovanich et al. describe a combination of cAMP-based affinity chromatography with quantitative mass spectrometry to investigate protein kinase A complexes in extracts of cells and tissues. De Jong et al. use activity-based chemical probes to profile the activity of the proteasome, which has recently emerged as an important cancer target, in cells and tissues. The next three chapters provide innovative protocols for the study of potential drug targets by chemical cross-linking and mass spectrometry. Mueller et al. provide a method to study protein–drug interactions, and Gasilova et al. employ cross-linking and MALDI-mass spec- trometry to study ligand modulation of protein–protein interactions. Jeon et al. provide a protocol for in vivo cross-linking via time-controlled transcardiac perfusion, which in prin- ciple enables the direct analysis of protein targets in animal models. The final set of chapters is concerned with small-molecule ligand and drug discovery. Casalena et al. describe the discovery of probe compounds by utilizing compound libraries immobilized on microarrays. Wolf et al. delineate general guidelines for working with small molecules, including aspects like storage, the preparation of solutions, and the determina- tion of solubility. The chapter by de Matos et al. provides guidelines for the use of the ChEBI database, which should be very helpful for researchers tasked with the selection of a particular probe or with building a small molecule collection to purpose. They describe the Chemical Entities of Biological Interest (ChEBI) database which helps to find probe molecules with the desired structural or biological features. Finally, many researchers will consider whether their research tool compound might have the potential to be developed into a drug. Zhang delivers a lucid analysis of the features that distinguish drugs from probe molecules, and lays out a set of rules for “drug likeness.” Affinity- and activity-based chemical probes, combined with quantitative immunodetec- tion and mass spectrometry techniques, are increasingly gaining appreciation as powerful strategies for the molecular analysis of complex biological systems in homeostasis and dis- ease. We hope that the methodologies described in this volume will contribute to a wider application of chemical proteomics methods in biochemical and cell biological laboratories. Heidelberg, Germany Gerard Drewes Marcus Bantscheff
  • 12. vii Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Part I Introduction 1 Mass Spectrometry-Based Chemoproteomic Approaches . . . . . . . . . . . . . . . . . . . . 3 Marcus Bantscheff 2 Chemical Proteomics in Drug Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Gerard Drewes Part II Small Molecules and Probe Design 3 Compound Immobilization and Drug-Affinity Chromatography . . . . . . . . . . . . . . 25 Uwe Rix, Manuela Gridling, and Giulio Superti-Furga 4 Affinity-Based Chemoproteomics with Small Molecule-Peptide Conjugates . . . . . . 39 Chaitanya Saxena 5 A Chemical Proteomic Probe for Detecting Dehydrogenases: Catechol Rhodanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Xia Ge and Daniel S. Sem 6 Probing Proteomes with Benzophenone Photoprobes . . . . . . . . . . . . . . . . . . . . . . 65 Akira Kawamura and Doina M. Mihai 7 Biotinylated Probes for the Analysis of Protein Modification by Electrophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Simona G. Codreanu, Hye-Young H. Kim, Ned A. Porter, and Daniel C. Liebler 8 Profiling of Methyltransferases and Other S-Adenosyl-l-Homocysteine- Binding Proteins by Capture Compound Mass Spectrometry . . . . . . . . . . . . . . . . 97 Thomas Lenz, Peter Poot, Elmar Weinhold, and Mathias Dreger Part III Target Discovery and Target Validation 9 Identifying Cellular Targets of Small-Molecule Probes and Drugs with Biochemical Enrichment and SILAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Shao-En Ong, Xiaoyu Li, Monica Schenone, Stuart L. Schreiber, and Steven A. Carr 10 Determination of Kinase Inhibitor Potencies in Cell Extracts by Competition Binding Assays and Isobaric Mass Tags . . . . . . . . . . . . . . . . . . . . 141 Carsten Hopf, Dirk Eberhard, Markus Boesche, Sonja Bastuck, Birgit Dümpelfeld, and Marcus Bantscheff 11 Affinity-Based Profiling of Dehydrogenase Subproteomes . . . . . . . . . . . . . . . . . . . 157 Xia Ge and Daniel S. Sem
  • 13. viii Contents 12 Probing the Specificity of Protein–Protein Interactions by Quantitative Chemical Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Duangnapa Kovanich, Thin Thin Aye, Albert J.R. Heck, and Arjen Scholten 13 Fluorescence-Based Proteasome Activity Profiling . . . . . . . . . . . . . . . . . . . . . . . . . 183 Annemieke de Jong, Karianne G. Schuurman, Boris Rodenko, Huib Ovaa, and Celia R. Berkers 14 Chemical Cross-Linking and High-Resolution Mass Spectrometry to Study Protein–Drug Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 Mathias Q. Müller and Andrea Sinz 15 Monitoring Ligand Modulation of Protein–Protein Interactions by Chemical Cross-Linking and High-Mass MALDI Mass Spectrometry . . . . . . . . . . . . . . . . . . 219 Natalia Gasilova and Alexis Nazabal 16 Time-Controlled Transcardiac Perfusion Crosslinking for In Vivo Interactome Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 Amy Hye Won Jeon and Gerold Schmitt-Ulms Part IV Ligand Discovery 17 Ligand Discovery Using Small-Molecule Microarrays . . . . . . . . . . . . . . . . . . . . . . 249 Dominick E. Casalena, Dina Wassaf, and Angela N. Koehler 18 Working with Small Molecules: Preparing and Storing Stock Solutions and Determination of Kinetic Solubility . . . . . . . . . . . . . . . . . . . . . . . . . 265 Andrea Wolf, Satoko Shimamura, and Friedrich B.M. Reinhard 19 A Database for Chemical Proteomics: ChEBI . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Paula de Matos, Nico Adams, Janna Hastings, Pablo Moreno, and Christoph Steinbeck 20 Working with Small Molecules: Rules-of-Thumb of “Drug Likeness” . . . . . . . . . . . 297 Ming-Qiang Zhang Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
  • 14. ix Contributors Nico Adams • Department of Genetics, University of Cambridge, Cambridge, UK Thin Thin Aye • Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University and Netherlands Proteomics Centre, Utrecht, The Netherlands Marcus Bantscheff • Cellzome AG, Heidelberg, Germany Sonja Bastuck • Cellzome AG, Heidelberg, Germany Celia R. Berkers • Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands Markus Boesche • Cellzome AG, Heidelberg, Germany Dominick E. Casalena • Chemical Biology Platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Steven A. Carr • Proteomics platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Simona G. Codreanu • Vanderbilt University School of Medicine, Nashville, TN, USA Annemieke de Jong • Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands Paula de Matos • European Bioinformatics Institute, Hinxton, UK Mathias Dreger • caprotec bioanalytics GmbH, Berlin, Germany Gerard Drewes • Cellzome AG, Heidelberg, Germany Birgit Dümpelfeld • Cellzome AG, Heidelberg, Germany Dirk Eberhard • Cellzome AG, Heidelberg, Germany Natalia Gasilova • CovalX AG, Schlieren, Switzerland; Department of Chemistry and Applied Biosciences, ETH, Zürich, Switzerland Xia Ge • Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI, USA Manuela Gridling • CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Janna Hastings • European Bioinformatics Institute, Hinxton, UK Albert J.R. Heck • Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University and Netherlands Proteomics Centre, Utrecht, The Netherlands Carsten Hopf • Cellzome AG, Heidelberg, Germany Amy Hye Won Jeon • Tanz Centre for Research in Neurodegenerative Diseases and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Akira Kawamura • Department of Chemistry, Hunter College of CUNY, New York, NY, USA Hye-Young H. Kim • Vanderbilt University School of Medicine, Nashville, TN, USA Angela N. Koehler • Chemical Biology Platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Duangnapa Kovanich • Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University and Netherlands Proteomics Centre, Utrecht, The Netherlands
  • 15. x Contributors Thomas Lenz • caprotec bioanalytics GmbH, Berlin, Germany Xiaoyu Li • Chemical Biology platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Daniel C. Liebler • Vanderbilt University School of Medicine, Nashville, TN, USA Doina M. Mihai • Department of Chemistry, Hunter College of CUNY, New York, NY, USA Pablo Moreno • European Bioinformatics Institute, Hinxton, UK Mathias Q. Müller • Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany Alexis Nazabal • CovalX AG, Schlieren, Switzerland Shao-En Ong • Proteomics Platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Huib Ovaa • Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands Peter Poot • Institute of Organic Chemistry, RWTH University, Aachen, Germany Ned A. Porter • Vanderbilt University School of Medicine, Nashville, TN, USA Friedrich B.M. Reinhard • Cellzome AG, Heidelberg, Germany Uwe Rix • CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Boris Rodenko • Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands Chaitanya Saxena • Shantani Proteome Analytics Pvt. Ltd., Pune, MH, India Monica Schenone • Proteomics platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Gerold Schmitt-Ulms • Tanz Centre for Research in Neurodegenerative Diseases and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Arjen Scholten • Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University andNetherlands Proteomics Centre, Utrecht, The Netherlands Stuart L. Schreiber • Chemical Biology Platform Chemical Biology Program, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Karianne G. Schuurman • Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands Daniel S. Sem • Department of Pharmaceutical Sciences, Concordia University Wisconsin, Mequon, WI, USA Satoko Shimamura • Cellzome AG, Heidelberg, Germany Andrea Sinz • Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany Christoph Steinbeck • European Bioinformatics Institute, Hinxton, UK Giulio Superti-Furga • CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Dina Wassaf • Chemical Biology Platform, The Broad Institute of MIT and Harvard, Cambridge, MA, USA
  • 16. xi Contributors Elmar Weinhold • Institute of Organic Chemistry, RWTH University, Aachen, Germany Andrea Wolf • Cellzome AG, Heidelberg, Germany Ming-Qiang Zhang • Merck Sharp Dohme (MSD), Chaoyang District, Beijing, PR, China
  • 19. 3 Gerard Drewes and Marcus Bantscheff (eds.), Chemical Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 803, DOI 10.1007/978-1-61779-364-6_1, © Springer Science+Business Media, LLC 2012 Chapter 1 Mass Spectrometry-Based Chemoproteomic Approaches Marcus Bantscheff Abstract The term “chemical proteomics” refers to a research area at the interface of chemistry, biochemistry, and cell biology that focuses on studying the mechanism of action of bioactive small molecule compounds, which comprises the mapping of their target proteins and their impact on protein expression and posttranslational modifications in target cells or tissues of interest on a proteome-wide level. For this purpose, a large arsenal of approaches has emerged in recent years, many of which employing quantitative mass spectrometry. This review briefly summarizes major experiment types employed in current chemical proteomics research. Key words: Affinity chromatography, Chemical proteomics, Drug profiling, Mass spectrometry, Posttranslational modifications, Quantitative proteomics, Stable isotope labeling, Targeted proteomics Experimental procedures commonly employed in chemical pro- teomics approaches can be categorized into two major groups (Fig. 1): (1) global proteomics approaches, aiming at the cell-wide characterization of cellular response to drug treatment, e.g., altered protein expression levels and posttranslational modifications; and (2) activity- or affinity-based protein profiling. The latter summa- rizing targeted chemoproteomic approaches which employ small molecular probes engineered to selectively capture protein targets/ subproteomes via a specific binding mode (e.g., by targeting co- factor binding sites) (1–8). 1. Introduction
  • 20. 4 M. Bantscheff For global chemoproteomic profiling, cells or animals are treated with a drug before system-wide proteome analysis to evaluate the cellular response in a global way (9–12). This strategy has become very attractive because of its simplicity and the unbiased nature of the analysis. Apart from cell permeability, there are no particular requirements for the small molecule compounds to be tested in such assays and the required quantitative mass spectrometric tech- niques are now available in many laboratories (for recent reviews, see refs. 13–16). However, since typically no protein enrichment is used, the changes that can be observed are limited by the analytical depth of the analysis and are often restricted to more abundant proteins. The analysis is further complicated by the fact that pro- teins found to be altered in abundance are not necessarily direct targets or downstream of the affected signaling cascade but often represent highly abundant proteins involved in stress response and/or housekeeping functions (12, 17). Hence, it is almost never possible to distinguish direct drug protein interactions from indi- rect effects. In examples illustrating the advantages and limitations 2. Global Chemoproteomic Profiling a b Wash Digest proteins LC-MS/MS Extract proteins Add Beads m/z Purify probes on beads Wash Digest proteins LC-MS/MS Extract proteins Add probe React probe c m/z Digest proteins LC-MS/MS Extract proteins m/z +Compound Control +Compound Control +Compound Control m/z m/z m/z 1 a.i. 1 a.i. 1 a.i. 1 a.i. 1 a.i. 1 a.i. Fig. 1. Experimental workflows in chemical proteomics. (a) Global proteomics approaches: Cells are treated with a com- pound, proteins are extracted from the sample, digested to peptides and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). (b) Activity-based protein profiling (ABPP) utilizes reactive probes specifically targeting the active site of an enzyme family. After protein extraction, the lysate is incubated with the probe to covalently attach to its targets. In the second step, probes and targets are purified using affinity chromatography before digestion and LC-MS/MS analysis. Pretreatment of cells with a small molecule compound binding to the active site of the investigated enzyme family leads to reduced capturing of the target enzyme via the reactive probes. (c) Alternatively, the compounds of interest can be modified and immobilized on a solid support. The immobilized drug is subsequently incubated with a cell extract to specifically enrich for target proteins that are subsequently identified by mass spectrometry. Competition with free excess inhibitor reduces the abundance of captured target proteins.
  • 21. 5 1 Mass Spectrometry-Based Chemoproteomic Approaches of global proteomics approaches, Chen and co-workers (12, 18, 19) evaluated the differential effect of the R- and S-enantiomers of atenolol, a β1-selective adrenoreceptor blocker, and the nonsteroi- dal antiinflammatory drug ibuprofen on two different cell types. The authors found 27 and 13 proteins to be differentially expressed, most of which can be classified as highly abundant (17). Yamanaka and co-workers applied a global proteomics approach for toxico- logical studies in animals (20). The authors studied the effects of 63 chemical compounds on protein expression in rat liver after 28 daily dosings and employed statistical methods to detect proteins characteristic for carcinogenicity. Subcellular and chromatographic fractionation allow for a more directed analysis of drug-induced changes in protein expression or posttranslational modifications. Lee and co-authors monitored his- tone modifications in response to treatment with histone deacety- lase (HDAC) inhibitors (10). In this study, human colon cancer cells were treated with HDAC inhibitors of varying degrees of selectivity followed by a simple prefractionation method to enrich for histone proteins. By employing label-free quantitative mass spectrometry, the authors identified HDAC-dependent histone acetylation patterns and quantified these in response to inhibitor treatment. Similarly, JmjD2A-dependent demethylation of K9 in histone H3 was monitored in response to cell treatment with pyridine-2,4-dicarboxylic acid derivatives (21). In several recent studies, chromatographic- or antibody-based enrichment methods have been employed to analyze kinase inhibitor-induced changes in protein phosphorylation on a global scale, thus mapping the net- work-level response to inhibitor treatment, and to infer signaling network topology (2, 11, 22, 23). A study by Mann and co-workers illustrated the impressive analytical depth that has been achieved in phosphoproteomics (22). Triple labeling SILAC (stable isotope labeling by amino acids in cell culture (24)) was used to analyze phosphorylation levels in growth factor-stimulated cells in the pres- ence or absence of kinase inhibitors. Among thousands of phospho- peptides, fewer than 10% were affected by the MAPK inhibitors U0126 and SB202190. By contrast, almost 1,000 phosphopeptides were affected by treatment of the leukemia cell line K562 with the potent but unselective BCR-Abl inhibitor dasatinib. A similar approach was applied to quantify changes in protein acetylation in response to the deacetylase inhibitors SAHA and MS-275 (25). In contrast to global approaches, recent developments in affinity- based proteomics techniques have enabled to directly determine protein binding profiles of small molecule drugs under close-to physiological conditions. These techniques are in principle based 3. Targeted Chemoproteomics Approaches
  • 22. 6 M. Bantscheff on affinity chromatography, typically using immobilized drugs or tool compounds (1, 8, 26) or covalent active site-labeling probes (3, 6, 27). Activity-based protein profiling (ABPP) via reactive probes designed to specifically bind to active sites of target enzymes was pioneered by the Cravatt laboratory. In this approach, the reactive probe is typically fused to an affinity tag, such as biotin, via a spacer. In a first step, the small molecule probe is incubated with the bio- logical sample and allowed to covalently attach to proteins it has affinity for. Subsequently, the formed probe–protein conjugates are captured using the affinity tag. Probes have been developed for a variety of enzyme classes including hydrolases (28), proteases (29, 30), kinases (31, 32), phosphatases (33), histone deacetylases (34), and glycosidases (35). For example, fluorescent activity-based probes were reported that enable substrate-free identification of inhibitors of uncharacterized enzymes. By using fluorescence polarization as a read-out, the approach is compatible to high- throughput screening with recombinant enzymes (36). A recent report demonstrated how ABBP can be used to screen compound libraries against an entire target class (37). The authors first synthe- sized a probe to selectively capture 80% of mammalian serine hydrolases, then screened 70 SHs against 140 structurally diverse carbamates and assessed the selectivity of hits using the very same approach. Patricelli and co-workers (31) used acyl phosphate- containing nucleotides, prepared from a biotin derivative and ATP or ADP to covalently modify ATP-binding proteins directly in cell extracts. Activity-based probes can be adapted for in situ and in vivo labeling by introducing a bio-orthogonal chemical handle, such as an alkyne. Probe-labeled enzymes are then captured by click chem- istry conjugation to azide-containing reporter tags (38–40). Probe design is a crucial step when developing ABPP-based assays and often requires detailed structural information to ensure selective binding of the probe to the catalytic site of target enzymes and to enable the efficient reaction of the active probe with suitable amino acid residues within or close to the catalytic site. In order to streamline probe development, a trifunctional probe design has been suggested consisting of a reversibly interacting selectivity group, a reactive group, and a sorting function (41). In this strategy, small molecules generating selectivity for individual enzyme classes are attached to a common building block containing reactive group and sorting function (i.e., biotin) that is common to all probes. Recent reports demonstrated examples for profiling of cAMP-binding pro- teins (42), kinases (43), and methyltransferases (44). For probes interacting with target enzymes with high affinity, however, probe design can be drastically simplified by omitting the reactive group and directly immobilizing the compound of interest on a solid support. The thus generated probe matrix is then incu- bated with cell extracts and mass spectrometry can be employed in
  • 23. 7 1 Mass Spectrometry-Based Chemoproteomic Approaches order to identify capture proteins (5, 45). Recent reports demonstrated successful applications of this approach for enrichment of protein kinases and characterization of binding profiles of kinase inhibitors (13, 23, 46–51), proteins binding to ATP/ADP (52), phosphati- dylinositols (53, 54), cyclic nucleotides (55, 56), histone deacety- lases (57), and peptides (58–60). Further, in several recent mode-of-action studies, immobilized analogs of lead compounds enabled target identification (61–64). Several large-scale studies have been performed for target class specific enrichment of kinase inhibitors using both reactive probes targeting the ATP binding pocket (31, 43) and immobilized unselective kinase inhibitors (46, 65) for affinity enrichment. The kinome coverage achieved with kinase inhibitor probes compared quite favorably to results obtained with reactive probes, indicating a limited impact of the bond-forming reaction with kinases on target class coverage. Qualitative binding profiles obtained in simple activity/affinity enrichment studies give only limited information about binding potencies of targets and off-targets detected. Consequently, the pharmacological relevance of detected off-target protein has to be validated using the standard repertoire of enzymatic assays (13, 47, 49, 50). While this might be feasible for very selective probes, cap- turing only few proteins, with recent mass spectrometric equip- ment often hundreds of proteins can be identified from a single affinity enrichment experiment. Further, the analysis of complex mass spectrometry data is impaired by the fact that the amounts of individual proteins captured do not represent the affinities of these proteins to the immobilized compound. Hence, additional evi- dence is required to distinguish low-abundant high-affinity inter- actors from low-affinity abundant ones, e.g., albumin and hemoglobin are known to have low affinity for a range of small molecules and many NADH/NADPH binding proteins bind to immobilized ATP-mimetics (13, 47, 49–51). In addition, proteins might bind to the resin or additional groups introduced to com- pounds for probe generation, e.g., linkers, reactive groups, biotin, etc. Some of these issues can be addressed by excluding those pro- teins from further analysis that have been frequently observed in independent experiments using different probe matrices (66). An elegant way to increase over-all specificity of the experiment is to design two probes/probe matrices, one containing the active com- pound of interest and one containing an inactive analog (45). Experiments with both matrices are then performed in parallel and candidate target proteins can be shortlisted upon differential display of results, e.g., using quantitative mass spectrometric methods. 4. Impact of Experiment Design and Quantitative Read-Out for Target Identification
  • 24. 8 M. Bantscheff However, inactive analogs are often not available and designing two independent probes is quite laborious. Utilizing reactive or immobilized analogs of a small molecule compound in order to identify its targets is a rather indirect approach and modifications introduced for probe generation might alter potency and selectivity of the compound under investigation. This limitation can be addressed by a competition-based experi- ment design. In this approach, probe/probe matrix binding is per- formed in the presence or absence of an excess of free, underivatized compound. Experiments are performed in parallel and quantitative mass spectrometry can be applied to identify those proteins for which captured amounts are strongly reduced in the presence of free compound as compared to experiments performed with vehi- cle control. In a recent application of this approach, Borawski et al. generated an affinity matrix consisting of a derivatized, bioactive PI4KB inhibitor linked to Sepharose beads used to purify cellular and/or viral proteins from a Huh7 HCV replicon cell lysate (61). To determine and quantify specific binding, the experiment was performed in a competition format by adding 10 μM PIK4B inhib- itor or DMSO alone into the replicon cell lysate prior to affinity purification. Bound proteins were eluted, digested with trypsin, and labeled with isobaric mass tags for relative and absolute quan- tification (iTRAQ, (67)) and combined prior to LC-MS/MS anal- ysis. This enabled to quantify binding displacement in the presence of free inhibitor relative to the vehicle. Several hundred proteins were identified and quantified in this experiment but only the bind- ing of class III PI4 kinases, PI4KA and PI4KB, was significantly reduced in the presence of free inhibitor. A similar, SILAC-based strategy was described by Ong et al. for the identification of targets of kinase inhibitors and imunophilin binders (68). Haystead and co-workers suggested a competition-based approach to estimate relative potencies of target proteins. ATP was linked to Sepharose-beads through the gamma phosphate group, thus generating a probe matrix selective for ATP-binding proteins including protein kinases as well as a variety of other proteins uti- lizing purine co-factors (52). After enrichment of ATP-binding proteins from cell extracts, the matrix was incubated with increas- ing concentrations of a set of antimalarial compounds and target proteins were eluted in a dose-dependent manner. In a variation of this approach, Patricelli and co-workers (31) used acyl phosphate- containing nucleotides, prepared from a biotin derivative and ATP or ADP to covalently modify ATP-binding proteins in the co-factor binding site. Biotinylated peptide fragments from labeled pro- teomes were subsequently captured and identified by mass spectrometry. Target kinases of kinase inhibitors were then deter- mined by comparing MS signals of probe-captured kinases in the absence and presence of excess free kinase inhibitors.
  • 25. 9 1 Mass Spectrometry-Based Chemoproteomic Approaches Stable isotope labeling-based quantitative mass spectrometry techniques for precise and accurate relative quantification of proteins (14, 16) have become an indispensable tool in chemical proteomic experiments, since identification of target proteins and their bind- ing affinities (IC50 s) largely depends on the ability to quantify differ- ences between vehicle control samples and samples incubated with different amounts of inhibitor. For example, iTRAQ labeling and LC-MS/MS were combined with a mixed kinase inhibitor probe matrix (“kinobeads”) for selectivity profiling of three inhibitors of the tyrosine kinase ABL developed for the treatment of chronic myeologeneous leukemia (CML); the phase II compound SKI-606 and the marketed drugs imatinib (Glivec) and dasatinib (46). In this study, cells or cell extracts were treated with inhibitor com- pounds at varying concentrations followed by incubation with the mixed kinase inhibitor matrix. Kinase inhibitors blocked the ATP binding pockets of target and off-target proteins as a function of concentration and affinity and consequently caused reduced abun- dance of these target proteins on the kinobeads matrix. Quantitative mass spectrometric analyses revealed binding profiles comprising 500 proteins including ~150 kinases. While dasatinib and SKI- 606 revealed very broad target profiles (46 and 42 proteins, respec- tively, showed 50% competition at 1 μM), imatinib was much more selective. In addition to the primary imatinib targets ABL/ BCR-ABL, ARG two novel target candidates, the receptor tyrosine kinase DDR1 (90 nM), and the quinone oxidoreductase NQO2 (43 nM) were identified and validated in independent studies (69, 70). Similarly, SILAC (24) has been employed in conjunction with kinase inhibitor matrices in order to determine the target profile of gefitinib (71). In a very recent report, such a chemoproteomics approach was applied to the analysis of inhibitors binding to native megadalton HDAC complexes. A total of 16 structurally diverse HDAC inhibitors were characterized for their selectivity in target- ing multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles, with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex, thus suggesting that the selectivity of HDAC inhibitors should be evaluated in the context of HDAC complexes rather than purified catalytic subunits (57). When cells are treated with an inhibitor, direct targets will be revealed by their reduced binding to the affinity matrix, however, e.g., protein kinases downstream of the respective target kinases will display an altered phosphorylation state due to the reduced signaling by the target kinase. For example, the case of imatinib- treated K562 cells, RSK1, and RSK3 were prominent examples for proteins exhibiting a significant downregulated phosphorylation state (46, 72).
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  • 30. 15 Gerard Drewes and Marcus Bantscheff (eds.), Chemical Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 803, DOI 10.1007/978-1-61779-364-6_2, © Springer Science+Business Media, LLC 2012 Chapter 2 Chemical Proteomics in Drug Discovery Gerard Drewes Abstract Real-world drug discovery and development remains a notoriously unproductive and increasingly uneconomical process even in the Omics era. The dominating paradigm in the industry continues to be target-based drug design, with an increased perception of the role of signaling pathways in homeostasis and in disease. Since proteins represent the major type of drug targets, proteomics-based approaches, which study proteins under relatively physiological conditions, have great potential if they can be reduced to practice such that they successfully complement the arsenal of drug discovery techniques. This chapter discusses examples of drug discovery processes where chemical proteomics-based assays using native endogenous proteins should have substantial impact. Key words: Chemical Proteomics, Drug target, Target deconvolution, Target validation, Drug discovery, Selectivity profiling Despite the dawn of the Omics era, drug discovery and develop- ment remains a notoriously unproductive and increasingly uneco- nomical process (1, 2). The dominating paradigm in the industry is still target-based drug design, with an increased perception of the role of signaling pathways in homeostasis and in disease (3). Because proteins represent the major type of drug targets, pro- teomics-based approaches, which allow to study a wide variety of proteins under relatively physiological conditions, have great potential if they can be reduced to practice such that they success- fully complement the arsenal of drug discovery techniques (4). Industry standard assays of drug action typically assess the biochemical activity of the purified target protein in isolation. Frequently, recombinant enzymes or protein fragments are used instead of the full-length endogenous proteins. The activity of a 1. Introduction
  • 31. 16 G. Drewes compound determined in this type of assays is often not predictive for its pharmacodynamic efficacy. One reason for this discrepancy is that an isolated recombinant protein, or protein fragment, does not necessarily reflect the native conformation and activity of the target in its physiological context, because of the lack of regulatory domains, expression of alternative splice variants, interacting regu- latory proteins, or incorrect protein folding or posttranslational modifications. As a consequence, data generated in such assays may not be predictive for the effects of a compound or drug in cell- based or in vivo models. Ideally, assays should be developed to generate data on native proteins in cell extracts or cell fractions, under conditions carefully optimized to preserve protein integrity, folding, posttranslational modification state, and interactions with other proteins. Both activity-based and affinity-based chemical proteomics techniques, as described in this volume, should com- plement, or in some instances replace the traditional recombinant protein-based assays. In target-based drug discovery, a project begins with the nomina- tion of a target. The target is typically defined as a protein which should be 1. Tractable: Its biochemical activity can be modulated by the desired therapeutic agent (e.g., a small molecule) in a dose- dependent fashion. 2. Validated: It mediates a pathophysiological process such that its modulation reverses a disease-relevant parameter, which can be measured in disease-related cell-based or animal models, and is expected to be predictive of human disease. Targets are often referred to as “druggable” and “clinically validated” when the modulation of the target was demonstrated to lead to the desired clinical outcome. Historically almost all drug- gable targets belong to a small number of target classes, biased toward cell surface proteins (e.g., G protein-coupled receptors, ion channels, or transporters) and a small number of intracellular protein classes (e.g., nuclear receptors, metabolic enzymes, kinases, or phosphodiesterases). A recent study estimated that the entirety of approved small molecule drugs acts through approximately 200 human proteins as targets (5), obviously a small number when compared to the 20,000–25,000 protein-coding genes in the human genome (6). It has been estimated that ten times as many suitable drug targets may exist, waiting to be discovered (7). In fact there are numerous proteins in pathways with a strong disease 2. Chemical Proteomics Can Aid More Informed Selection and Validation of Targets
  • 32. 17 2 Chemical Proteomics in Drug Discovery implication, e.g., based on pathobiochemical and human genetic evidence, which are not tractable by current small molecule-based approaches. Chemical proteomics approaches should serve to expand the number of accessible drug targets by aiding the identi- fication of tractable targets without the heavy bias toward the traditional target classes. This type of “target deconvolution” approaches was pioneered by the Schreiber laboratory in the clas- sical studies which identified the molecular targets of immunosup- pressants (8, 9). More recent exemplary approaches employed a combination of screening of diverse compound libraries in cell- based assays, which are not biased toward a particular family of targets, with chemoproteomics-based target identification. Huang et al. discovered the tankyrase proteins as tractable targets in the Wnt signaling pathway, which plays a central role in colon cancer but was characterized by a dearth of tractable drug targets (10). Using a related strategy, Fleischer et al. found that the potent and selective cytotoxic agent CB30865 exerts its effects by inhibition of nicotinamide phosphoribosyltransferase, an enzyme in the NAD biosynthetic pathway which helps cancer cells to sustain their increased energy metabolism (11). In another recent study, cell- based screening was performed for the upregulation of apolipopro- tein AI production, and the proteomic profiling of hit compounds led to the unexpected discovery of bromodomain proteins as tractable targets for the modulation of the expression of apolipo- protein AI and certain proinflammatory genes (12). These bro- modomain inhibitors exhibit a novel mechanism of action by blocking a protein–protein interaction formed between acetylated histones and BET-family bromodomains, which were not previ- ously regarded as tractable targets. These and other successful studies support the notion that there is a general need for small molecules as research tools to study protein function, particularly for proteins which are not classical drug targets. Both the Structural Genomics Consortium (http:/ /www.thesgc.org) and the Center for Protein Research (http:/ /www.cpr.ku.dk) have recently initi- ated extensive programs for the development of chemical probes which will be made available to the scientific community. Many drug discovery assays rely on the ability to express and purify the target protein in active form in the substantial amounts – typically milligrams of pure protein – necessary for the screening of com- pound libraries. The drug industry has encountered many so-called “difficult” target classes where this is not easily achieved, for instance, because the target protein is very large or requires addi- tional factors like interacting proteins for proper activity. Therefore, 3. Chemical Proteomics-Based Screening of Compound Libraries
  • 33. 18 G. Drewes methods based on immobilized probe compounds to capture the target directly from a cell or tissue extract without further purifica- tion can represent a viable alternative strategy. This approach was used by Fadden et al. who captured purine-binding proteins from porcine tissue with ATP-derivatized Sepharose and performed affinity elutions with 5,000 different compounds, resulting in the identification of 463 small molecule compounds eluting a total of 77 distinct proteins. Among these, novel and structurally diverse inhibitors of the cancer target Hsp90 were identified, which were further optimized to enter clinical development (13). A different strategy was used by Bantscheff et al. who screened a compound library for histone deacetylase (HDAC) inhibitors in a human cell line extract, using an immobilized hydroxamate-based probe. Here, compounds were added directly to the cell extract rather than using them for elution, such that each compound was assayed for the inhibition of the binding of HDACs to the immobilized probe (14). An important feature of both approaches is that the entire complement of proteins binding selectively to the immobi- lized probe is screened simultaneously. This represents a major advantage over traditional screening approaches, in particular, for target classes with a substantial number of structurally related tar- gets, like protein kinases or deacetylases, because possible “off- targets” (undesired additional proteins, which typically share a related active site with the target) are revealed early in the project. In conventional approaches, one is left to resort to educated guesses regarding possible “off-targets,” and distinct assays have to be con- figured for each individual protein. Despite the fact that drugs are usually optimized against a single target, many compounds exhibit polypharmacology, i.e., they act on multiple targets. These “off-targets” can increase the therapeu- tic potential of a drug, but they might also cause toxic side effects, which represent a major reason why drugs fail in clinical develop- ment (15). An important recent example was the chemoproteomics- based identification of cereblon (CRBN) as a target of the drug thalidomide which mediates the drug’s teratogenic effects (16). However, for oncology drugs, polypharmacology is the rule rather than the exception, as they often target proteins from large target classes with a high degree of structural conservation around the active site, like protein and lipid kinases, HDACs, or heat shock proteins. Compared to a truly selective drug, such a spectrum of targets is more likely to produce toxic side effects, but in oncology the increase in therapeutic potential may outweigh this disadvan- tage (17). Conventional strategies typically rely on assay panels comprising 10–100 purified enzymes to address compound 4. Chemical Proteomics for Drug Target Profiling
  • 34. 19 2 Chemical Proteomics in Drug Discovery potency, selectivity, and potential off-target liabilities (18). The recent progress in affinity-based proteomic techniques has enabled the direct determination of protein-binding profiles of small mol- ecule drugs under close to physiological conditions. These tech- niques utilize immobilized compounds as noncovalent affinity baits (14, 19–22) or covalent active-site labeling probes (23, 24). The affinity probes are designed to selectively enrich a larger set of up to several hundreds of proteins defined by structurally related active sites, which can be viewed as chemically tractable subproteomes (25). Noncovalent probes are used either immobilized to an affin- ity matrix like sepharose or conjugated to biotin, and have been used successfully for purine-binding proteins (26), protein kinases including transmembrane receptor kinases (21, 22), lipid kinases (27, 28), phosphodiesterases (29), and HDACs (14). Covalent active-site labeling probes are typically biotin conjugates and have been applied to kinases (30), GTPases (31), methylases (32), dehy- drogenases (33), serine-, cysteine-, metallo-, and proteasomal pro- teases (23, 34, 35), and HDACs (36). These methodologies typically generate protein affinity profiles for the immobilized com- pounds, which may reveal novel target candidates, but precautions must be taken to avoid false positives due to the background prob- lems caused by nonspecific interactions with abundant proteins. Moreover, for the application to drug discovery, e.g., in screening or affinity/selectivity profiling assays, the generation of robust quantitative data for hit and lead compounds is an absolute neces- sity. These problems can be managed if the affinity capture proto- cols are formatted as quantitative competition-binding assays. This can be achieved by adding the compound of interest in its free form in the tissue extract, before or together with the affinity matrix or the active site label, such that the free compound binds to its targets in the lysate, thereby effectively competing with the capturing probe. By assaying the free compound in the cell extract over a range of concentrations, dose–response binding curves are generated for as many proteins as can be captured by the probe compound and robustly quantified. In case of the “Kinobeads” matrix for protein kinases and the hydroxamate matrix for HDACs developed by Bantscheff et al., more than 1,000 proteins were found to bind to the matrix and were routinely quantified in drug- profiling experiments using a competition binding assay format coupled to protein quantification by isobaric tagging and high- resolution LC-MS/MS peptide sequencing (14, 22). For a more detailed discussion of qualitative and quantitative small molecule target profiling, the reader is referred to recent comprehensive reviews (20, 23, 37). Finally, in addition to the in vitro applications described above, many chemical proteomics strategies can poten- tially be adapted to the identification and activity profiling of tar- gets in living cells and in animal models (38). In conclusion, the recent advances in chemical proteomics and in analytical instrumentation have promoted new drug discovery
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  • 37. Part II Small Molecules and Probe Design
  • 38. 25 Gerard Drewes and Marcus Bantscheff (eds.), Chemical Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 803, DOI 10.1007/978-1-61779-364-6_3, © Springer Science+Business Media, LLC 2012 Chapter 3 Compound Immobilization and Drug-Affinity Chromatography Uwe Rix, Manuela Gridling, and Giulio Superti-Furga Abstract Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to under- standing its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identifica- tion of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobiliza- tion, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatogra- phy step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry. Key words: Drug, Target, Affinity chromatography, Immobilization, Chemical proteomics, Mechanism of action Promiscuity of small molecule drugs and tool compounds has important implications for understanding their cellular mecha- nisms, side effects, and clinical potential. Chemical proteomics is a postgenomic, mass spectrometry (MS) and bioinformatics-enabled version of previous versions of drug-affinity chromatography that can identify a drug’s cellular target profile, thus aiding in the subsequent 1. Introduction
  • 39. 26 U. Rix et al. characterization of its molecular mechanism of action (1, 2). This approach requires the immobilization of the compound of interest on solid support as an early step. The strategy for immobilization has to be designed very carefully as chemical modification of any bioactive compound harbors the risk of impairing or abrogating its biological activity through disruption of the protein interaction interface. Therefore, one should utilize all available structure– activity relationship information, which can, for instance, be obtained by activity studies with structural analogs or from X-ray co-crystal structures. If such information is unavailable, it is recom- mended to immobilize the bait compound in separate experiments via two or more different linker attachment points. In some cases, when a cellular or biochemical readout is at hand, these analogs can be tested for retention of activity. When employing cellular assays, though, it has to be kept in mind that modifying a compound might also alter its cell permeability properties. In addition, some molecules are sensitive to various commonly employed conditions, such as acid, base, or heat. Therefore, any broadly applicable modification or immobilization reaction has to be efficient and mild. This is the case for several different reactions employing, e.g., epoxide or amide chemistry (3). The formation of an amide bond from an activated N-hydroxy-succinimide (NHS) ester and the amino group (primary or secondary) of a bioactive compound (analog) is readily achieved at room temperature and the reaction usually comes to completion within several hours (4). Compounds containing hydroxy- or carboxy-groups can be conveniently converted into amines by Steglich esterification with Boc- or Fmoc-protected amino acids or mono-protected ethylenediamine, respectively, and subsequent deprotection (5). The choice of protecting group should be based on chemical stability of the bait compound under deprotection conditions (6). Other important considerations for drug-affinity chromatography are the choice of buffer and pH. Just as critical is the efficient inhibition of cellular proteases, which would destroy proteins and prevent affinity enrich- ment, and possibly enzymes that carry out unscheduled posttrans- lational modifications in vitro (e.g., protein dephosphorylation), as well as the choice and concentration of detergent, which aids in the solubilization of proteins. In the following, we present the detailed workflow for immobilization and affinity chromatography, which we have previously applied to the characterization of several clinical kinase inhibitors by chemical proteomics (7). We exemplify the approach with a study on the BCR-ABL tyrosine kinase inhibitor dasatinib (BMS-354825, trade name Sprycel) (8), a drug in clini- cal use for chronic myeloid leukemia (CML) and several other malignancies. In this example, drug-affinity chromatography is performed using the CML cell line KU812 (9).
  • 40. 27 3 Compound Immobilization and Drug-Affinity Chromatography 1. NHS-activated Sepharose 4 Fast Flow (GE Healthcare) (see Note 1). 2. Dimethyl sulfoxide, absolute over molecular sieve (DMSO, Fluka). 3. Isopropanol, absolute over molecular sieve (iProp, Fluka). 4. Triethylamine (TEA, Sigma). 5. Ethanolamine (Aldrich). 6. 2 mL microcentrifuge tubes (Eppendorf). 1. Methanol (MeOH), LC-MS grade (Fisher Scientific GmbH). 2. Isopropanol (iProp), LC-MS grade (Fisher Scientific GmbH). 3. Water, LiChrosolv grade (Merck). 4. Formic acid. 5. Chromatography column: SunFire C18 , 5 μm, 3.0×50 mm (Waters). 6. HPLC 2795 (Waters). 7. Photo diode array detector 2996 (Waters). 8. ZQ 2000 single quadrupole mass spectrometer (Waters/ Micromass). 1. Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) sup- plemented with 10% fetal bovine serum (Gibco) and penicillin/ streptomycin 100× (PAA Laboratories GmbH). 2. Cell culture dishes, Ø 150 mm (VWR International). 3. 4× Concentrated Laemmli sample buffer containing 10% β-mercaptoethanol. 4. Lysis buffer 1×: 50 mM Tris–HCl, 100 mM NaCl, 0.2% NP-40, 5% glycerol, 1.5 mM MgCl2 , 25 mM NaF, 1 mM Na3 VO4 , 1 mM phenylmethylsulfonyl fluoride, 1 mM dithio- threitol (DTT), 10 μg/mL TLCK, 1 μg/mL leupeptin, 1 μg/ mL aprotinin, and 10 μg/mL soybean trypsin inhibitor (Sigma), pH 7.5 (see Note 2). 5. γ-Globin (1 μg/μL) (Bio-Rad). 6. Protein Assay Dye Reagent Concentrate (5×) (Bio-Rad). 7. Hypodermic Needle, 20 G×1½ (Terumo® ), single-use syringe 10 mL (B. Braun Melsungen AG). 8. Cuvettes half-micro, 2.5 mL, PMMA (VWR International). 9. Polycarbonate ultracentrifuge tubes (Beckman Coulter). 10. Ultracentrifuge and fixed-angle rotor (Beckman Coulter). 2. Materials 2.1. Coupling to Solid Support 2.2. LC-MS Analysis of the Coupling Reaction 2.3. Cell Culture, Harvest, Lysis, and Protein Quantification
  • 41. 28 U. Rix et al. 1. Lysis buffer 1× (see above). 2. 4× Concentrated Laemmli sample buffer (containing 10% β-mercaptoethanol) (see above). 3. Polycarbonate ultracentrifuge tubes (Beckman Coulter). 4. Spin columns Mobicol (MoBiTec GmbH). 5. Spin column lower filters – 90 μm pore size (MoBiTec GmbH). 6. 1.5 and 2 mL microcentrifuge tubes (Eppendorf). 1. Iodoacetamide (13 μg/μL) (Sigma). 2. Mini-PROTEAN Tetra electrophoresis system (Bio-Rad Laboratories). 3. 4× Running gel buffer (SDS buffer): 1.5 mM Tris–HCl and 0.54% SDS, pH 8.8. 4. 4× Gel buffer 2: 2 M Tris, 1.6 mM SDS, pH 6.8. 5. Ammoniumperoxide sulfate (APS), acrylamide, tetramethyl- ethylenediamine (TEMED, Sigma). 6. 5× Running buffer: 602 g Tris, 2,880 g glycine, 200 g SDS, 20 L water, pH 8.3. Do not use HCl! 7. Broad range 7–175 kDa prestained protein marker (New England Biolabs). 1. Fixing buffer: 40% ethanol, 10% acetic acid. 2. Washing buffer: 35% ethanol or distilled water. 3. Sensitizing buffer: 0.02% Na2 S2 O3 . 4. 0.2% AgNO3 solution (refrigerated). 5. Developing buffer: 3% Na2 CO3 , 0.05% formaldehyde. 6. Quenching solution: 5% acetic acid. 1. 10× Western blot buffer: for 2 L dissolve 60.7 g Tris and 288.4 g glycine in distilled water. 2. 1× Western blot buffer: 10% (v/v) 10× Western blot buffer, 10% (v/v) methanol. 3. Wash buffer: PBS with 1% Tween-20 (PBS-T). 4. Blocking buffer (in this example, see Note 3): 3% bovine serum albumin (BSA) in PBS-T. 5. Primary antibody dilution (in this example): 1:2,000 mono- clonal mouse anti-ABL (21–63) (Santa Cruz Biotechnology) in 3% BSA/PBS-T (see Note 4). 6. Secondary antibody dilution (in this example): 1:2,000 horse radish peroxidase (HRP)-conjugated anti-mouse (Rockland Immunochemicals) in PBS-T. 2.4. Affinity Pulldown 2.5. One-Dimensional SDS-PAGE 2.6. Silver Staining 2.7. Immunoblot
  • 42. 29 3 Compound Immobilization and Drug-Affinity Chromatography 7. Amersham ECL Plus™ Western Blotting Detection Reagents (GE Healthcare). 8. Nitrocellulose transfer membrane (Protran BA 85, 300 mm×3 m, 0.45 μm). 9. Saran wrap. 10. Amersham Hyperfilm ECL (GE Healthcare). 11. Hoefer Semi-Phor semi-dry transfer unit (Hoefer, Inc.). This section is illustrated in Fig. 1. 1. The beads are provided in a slurry of approximately 50% iso- propanol. For one pull down, pipette 100 μL of this slurry into a 1.5 mL microcentrifuge tube (see Note 5). Compare the settled bed volume with 50 μL DMSO. Add or remove slurry until 50 μL bed volume is reached (see Note 6). 3. Methods 3.1. Coupling to Solid Support Fig. 1. Immobilization strategy for the BCR-ABL kinase inhibitor dasatinib. The “couple- able” analog c-dasatinib was designed based on the X-ray co-crystal structure of ABL with dasatinib, which indicated that the hydroxy group extends into the solvent space (10). The dasatinib affinity matrix is generated via coupling of c-dasatinib to NHS-activated sepharose.
  • 43. 30 U. Rix et al. 2. Centrifuge the beads for 3 min at room temperature with 75×g and remove the supernatant. 3. Add 50 μL of DMSO, resuspend gently by inverting several times, centrifuge (as before), and discard the supernatant. Repeat this washing step three times with 0.5 mL DMSO. Then resuspend beads in 50 μL DMSO. 4. Dissolve bait compound to a final concentration of 10 mM in DMSO and add 5 μL of this stock solution and 0.75 μL TEA to the 50% bead slurry, mix carefully and incubate on roto- shaker for 16–24 h (but at least for 8 h) at room temperature with 10 rpm (see Note 7). 5. Centrifuge the beads (as before) and remove 10 μL (»5 nmol) of the supernatant for the LC-MS control. This is the T16 time point for the coupling control. 6. If the drug was successfully coupled to the beads, block the unreacted NHS-ester groups by adding 25 μL ethanolamine and incubate on roto-shaker for further 16–24 h (but at least for 8 h) at room temperature with 10 rpm. 7. Centrifuge beads, remove the supernatant, wash twice with 0.5 mL of DMSO, and discard the supernatant again. 8. Either proceed directly with the drug pulldown and wash the beads with Lysis buffer or add 50 μL isopropanol, centrifuge, discard the supernatant, and repeat this washing step twice with 0.5 mL isopropanol each. The beads can be stored in iso- propanol at 4°C (away from light) (see Note 8). This section is illustrated in Fig. 2. 1. Remove 0.5 μL of a 10 mM bait compound stock solution and add 9.5 μL DMSO. This is the T0 time point. Add 10 μL MeOH to each sample (T0 and T16 ). 2. Inject 5 μL of the samples beginning with the T16 time point to avoid carryover. 3. The applied solvent system and LC conditions were: (a) Solvent A=0.1% formic acid in H2 O (b) Solvent B=80%MeOH/20% iProp (c) Flow rate=0.5 mL/min (d) 0–6 min 90% A/10% B to 100% B (linear gradient, curve 6) (e) 6–8 min 100% B (f) 8–8.5 min 100% B to 90% A and 10% B (linear gradient, curve 6) (g) 8.5–11 min 90% A and 10% B 3.2. LC-MS Analysis of the Coupling Reaction
  • 44. 31 3 Compound Immobilization and Drug-Affinity Chromatography 4. The column temp was 40°C. 5. The reaction was analyzed on a photodiode array detector (PDA) scanning a range of wavelengths between 210 and 500 nm. 6. The MS was set to scan a mass/charge ratio from 100 to 1,000, switching continuously between the positive and negative elec- trospray ionization (ESI) mode. 7. The MS parameters were: (a) Capillary voltage: 3.2 (ESI+/−) (b) Cone voltage: 32 (ESI+), 30 (ESI−) (c) Extractor voltage: 3 (ESI+), 4 (ESI−) (d) RF lens voltage: 0.3 (ESI+), 1.2 (ESI−) (e) Source temperature: 150 (ESI+/−) (f) Desolvation temperature: 400 (ESI+/−) (g) Desolvation gas (N2 ): 450 (ESI+/−) (h) Cone gas (N2 ): 50 (ESI+/−) (i) HM and LM resolution: 15 (ESI+/−) (j) Ion energy: 0.6 (ESI+) and 0.1 (ESI−) Fig. 2. LC-MS analysis of immobilization reaction of c-dasatinib. (a) UV chromatograms of T0 and T16 samples measured at 254 nm. The peak at a retention time of 3.00 min disappears after 16 h of incubation. (b) Positive mode electrospray MS spectra at 3.00 min retention time of the T0 and T16 samples confirming that the observed peak represents c-dasatinib, which completely disappears after incubation, i.e., couples to the activated matrix.
  • 45. 32 U. Rix et al. 1. Culture KU812 cells in 20 mL DMEM on a 15 cm dish. After cells approach confluence, centrifuge the cell suspension for 3 min with 300×g. Aspirate the medium and resuspend cells in 2 mL fresh medium. 2. Fill 20 mL DMEM in four new 15 cm dishes and add 0.5 mL of the cell suspension. After ca. 2 days cells approach conflu- ence and then need to be split again. 3. To harvest the cells, centrifuge the cell suspension, aspirate the medium, and wash with PBS. Shock freeze the samples in liq- uid nitrogen and store them at −80°C until further use or use them directly for lysis and the protein quantification. 1. Thaw pellet on ice and resuspend in appropriate amount of Lysis buffer (depending on pellet size) and pull it through a 0.9 mm syringe ten times (see Note 9). 2. Transfer the homogenate to a 15 mL Falcon tube and incubate on ice for 30 min. 3. Transfer lysate to an ultracentrifuge tube, balance tubes, and centrifuge for 10 min at 4°C with 20,000×g. 4. Transfer the supernatant to a fresh polycarbonate ultracentri- fuge tube, balance tubes, and centrifuge for 1 h at 4°C with 100,000×g. 5. Transfer supernatant to a fresh 15 mL Falcon tube and keep on ice. 6. Determine the protein concentration by the Bradford method using a freshly determined standard curve. Apply 0, 5, 10, 15, 20, 25, and 30 μL γ-globin and 0.5, 1, and 2 μL lysate to the cuvettes. Add 1 mL of the dye reagent (1×) to each cuvette, mix by careful vortexing, and measure the absor- bance at 595 nm. 7. Prepare 25 mg aliquots in microcentrifuge tubes. Use the lysates directly for the drug pulldown or shock freeze the sam- ples in liquid nitrogen and store them at −80°C until use. 1. Prepare the 1× Lysis buffer freshly and keep it on ice. 2. Dilute the thawed cell lysate (preferably, prepare cell lysate freshly) with 1× Lysis buffer to a total volume of 3 mL (for 25 mg total protein) transfer to Beckman ultracentrifuge tube, balance tubes, and centrifuge for 20 min at 4°C with 100,000×g. 3. Collect “total cell lysate” (TCL) sample (100 μg per Western blot), add the appropriate amount of 4× Laemmli sample buf- fer, and denature proteins at 100°C for 3–5 min. 4. Pipette 100 μL 50% drug-bead slurry (i.e., 50 μL settled bead volume=0.1 μmol drug) into a 2 mL Microcentrifuge tube 3.3. Cell Culture and Harvest 3.4. Preparation of Total Cell Lysate and Protein Quantification 3.5. Affinity Pulldown
  • 46. 33 3 Compound Immobilization and Drug-Affinity Chromatography (always use cut pipette tips for pipetting beads) and centrifuge beads for 3 min at 4°C with 75×g, remove supernatant. 5. Add 1 mL 1× Lysis buffer and wash beads by gently resuspend- ing and inverting several times, centrifuge beads, remove super- natant, repeat wash step three times, and remove supernatant. 6. Combine and resuspend the beads with the centrifuged cell lysate. 7. Incubate on roto-shaker for 2 h at 0–4°C with 10 rpm. 8. Centrifuge beads, remove and discard supernatant, but leave ca. 700 μL buffer in microcentrifuge tube (perform following steps in cold room at 0–4°C). 9. Resuspend beads gently and transfer to (plugged!) Mobicol columns, let beads settle by gravity. 10. Drain remaining buffer by gravity flow, fill with fresh 1× Lysis buffer and connect to 30 mL syringe with luer lock tip. 11. Add 7.5 mL 1× Lysis buffer and let the buffer drain by gravity flow (possibly apply gentle pressure with syringe plunger). 12. Place the column in a 2 mL microcentrifuge tube and centri- fuge for 1 min at 4°C with 100×g, plug the column, and place it in a 1.5 mL microcentrifuge tube. 13. Add 30 μL 4× Laemmli sample buffer and denature proteins at 100°C for 3–5 min. 14. Open first the top lid of the column, then unplug the bottom (careful!) and place back into the microcentrifuge tube. 15. Centrifuge for 1 min at room temperature with 400×g, collect eluate (this is the first elution sample) and replug the column. 16. Place column in another 1.5 mL microcentrifuge tube, add 30 μL 4× Laemmli sample buffer and denature proteins at 100°C for 3–5 min. 17. Unplug the column, centrifuge (as before) and collect eluate (this is the second elution sample) (see Note 10). 18. Store at −20°C until SDS-PAGE analysis. 1. For the 7% separating gel, mix 2.5 mL SDS buffer, 2.35 mL 30% acrylamide, 5.17 mL H2 O, 100 μL 10% APS, and 10 μL TEMED and fill ca. 4 mL (leave space for 2.5 mL of stacking gel) of the resulting solution between the two glass plates and overlay with 1 mL isopropanol, wait until the gel has polymer- ized (ca. 20 min) and pour off the isopropanol. 2. For the stacking gel, mix at first 25 mL Gel buffer 2 with 16.6 mL 30% acrylamide and 58.4 mL H2 O. Take 10 mL of this solution and add 100 μL 10% APS and 15 μL TEMED. Pour ca. 2.5 mL on the polymerized separating gel and insert a comb. 3.6. One-Dimensional SDS-PAGE
  • 47. 34 U. Rix et al. 3. After another 20 min remove the comb and place the gel in the electrophoresis chamber, which is filled with running buffer. 4. For immunoblotting, load 10 μL (5 μl each of first and second elution) per well, in the first well inject 5 μL of the protein marker. For silver staining, mix 10 μL of the first and second elutions and alkylate the sample with iodoacetamide (final con- centration 13 μg/μL) for 20 min in the dark. Then load the samples on the gel. If possible, fill the first and the last well of the gel with 8 μL of Laemmli sample buffer to ensure an even running profile. 5. Complete the assembly of the gel unit and connect the power supply. The gel can be run at 120 V or higher, if time is short. When the lysates are run through the gel it can be transferred to the nitrocellulose membrane or used for silver staining. This section is illustrated in Fig 3a. 1. Perform all the following steps in a dust-free cabinet to mini- mize keratin contamination. First, fix the gel with 40% ethanol and 10% acetic acid (always add sufficient solution to com- pletely cover the gel) for 1 h at room temperature on a rocker or at 4°C overnight. 2. Wash the gel two times with 30% ethanol for 20 min and for another 20 min with water (pro analysi). In the meantime prepare the sensitizer, the silver nitrate (refrigerator) and the developer. 3.7. Silver Staining Fig. 3. SDS-PAGE-based analysis of the dasatinib pulldown eluate from KU812 CML cells (9). (a) Silver staining shows several bands that subsequent LC-MS/MS analysis reveals to correlate with validated dasatinib target kinases. (b) Immunoblot with anti-ABL 21–63 antibody shows the enrichment of BCR-ABL and c-ABL by the dasatinib affinity matrix while an ampicillin control matrix does not display affinity for the dasatinib cognate targets. SFK SRC family kinase.
  • 48. 35 3 Compound Immobilization and Drug-Affinity Chromatography 3. Sensitize the gel with 0.02% Na2 S2 O3 for 1.5 min and wash it three times for 20 s with water (pro analysi). 4. Incubate the gel with refrigerated 0.2% AgNO3, for 20 min. 5. Discard the silver nitrate in heavy metal waste and wash the gel again (three times for 20 s). Transfer the gel into a clean Petri dish after the second wash step to keep the background as low as possible. 6. Develop the gel in 3% Na2 CO3 and 0.05% formaldehyde. Rock the Petri dish until the solution changes to a yellow color. Remove the liquid and allow the development without liquid. If the staining is not sufficient, repeat the developing step. 7. When all the bands are visible on the gel, quench the developer by removing the liquid from the Petri dish and adding 5% ace- tic acid. Leave the gel in the acetic acid for a minimum of 5 min. 8. Remove the acid, place the gel in the lid of the Petri dish, cover it with parafilm, and scan the developed gel for the documen- tation using a conventional picture-scanner. 9. Cover the gel with water if the gel is stored in the refrigerator for further analysis. This section is illustrated in Fig. 3b. 1. Take three 8×9 cm filter papers, soak them in 1× Western blot buffer (containing 10% methanol), and place them in the transfer unit. Then take a 7×8 cm membrane, soak it in the buffer, and place it on the filter paper. Cut away the comb of the gel, soak the gel in the buffer, carefully lay it on top of the membrane, and cover it with another three soaked filter papers. Finally ensure that there are no air bubbles in the resulting sandwich and close the transfer cassette. The transfer can be accom- plished at 56 mA (1 mA per cm²) for 1.5 h. 2. Incubate the membrane for 1 h in approximately 50 mL Blocking buffer. 3. Discard the Blocking buffer and incubate the membrane with the primary antibody dilution over night in the cold room. Use gentle agitation. 4. Wash the membrane three times for 5 min with PBS-T. 5. Add 10 mL of the secondary antibody dilution (always use a fresh solution) and incubate for 1 h. 6. Discard the solution and wash the membrane again three times for 5 min. 7. Mix together the ECL Plus solution (1:40) and cover the membrane with 2 mL of the resulting solution. Incubate for 4 min and let the redundant ECL solution drip down. Envelope the membrane with the Saran-Film (ensure that there are no 3.8. Immunoblot
  • 49. 36 U. Rix et al. bubbles) and place it in an X-ray film cassette. Perform the remaining steps in a dark room under safe light conditions. 8. Insert a developing film in the cassette and expose the film for a few seconds to minutes (see Note 11). 1. Always use cut pipette tips for pipetting beads with a clean scalpel or scissors. 2. Use high-purity distilled water of consistent quality to avoid contaminations, such as DNase and RNase free distilled water (Gibco). 3. The primary antibody and therefore the blocking conditions depends on any prior knowledge of validated target proteins of the bait compound. For our example, we have chosen dasat- inib as the bait compound, the cognate target of which is ABL. Therefore, we will delineate in the following conditions for the anti-ABL (21–63) antibody (Santa Cruz Biotechnology). 4. The primary antibody can be often reused for subsequent applications. If used in milk/PBS-T, it should be stored at 4°C and be used within a few days. If used in BSA, it can usually be stored at −20°C for several months. 5. Always use aerosol filter pipette tips to avoid cross contamina- tion of samples, as mass spectrometers are highly sensitive instru- ments, which will detect even trace amounts of contaminants. 6. Contamination of samples with keratin is one of the most com- mon and serious complications for proteomics mass spectrom- etry analysis, due to the abundance of this family of proteins and dynamic range limits of mass spectrometers. There are sev- eral sources of keratin contamination, such as dust, hair, skin, and clothing. It is therefore essential to take the precautions to reduce keratin levels as much as possible. (a) Always wear gloves and laboratory coat as much for per- sonal protection as for avoiding keratin contamination. (b) Avoid wearing woollen clothing, but rather synthetics or cotton. (c) Use a designated dust-free clean bench. (d) Use a dedicated set of pipetting and electrophoresis equipment. (e) Prepare buffers and reagents freshly from designated stock solutions. 7. The bait compound needs to contain either a primary or sec- ondary amino group, as does c-dasatinib, which was designed 4. Notes
  • 50. 37 3 Compound Immobilization and Drug-Affinity Chromatography based on the available X-ray co-crystal structure of dasatinib with ABL (10). Alternatively, primary or secondary alcohols can be readily esterified under Steglich conditions, i.e., in the presence of dimethylaminopyridine (DMAP) and dicyclohexy- lcarbodiimide (DCC) (5). For example, the hydroxyl group of dasatinib can be esterified with N-Boc-glycine. Subsequent removal of the Boc protecting group with trifluoroacetic acid yields a primary amine suitable for immobilization. 8. Duration of storage depends strongly on the stability of the bait compound. Although the dasatinib matrix was sufficiently stable, our experience with other compounds suggests that a storage of maximum 2 weeks should not be exceeded. If pos- sible, drug beads should be used immediately. 9. Some primary cells express high levels of protease activity which may require the addition of additional protease inhibi- tors to the Lysis buffer, such as the Complete Protease Inhibitor Cocktail Tablets (Roche). 10. Samples from the first and second elution are stored separately, but can be combined directly before loading on SDS-PAGE gel, if desired. 11. Exposure time typically varies. Acknowledgements This work was supported by the Austrian Federal Ministry for Science and Research (BMWF) under the GEN-AU program (GZ BMWF-70.081/0018-II/1a/2008) and the Austrian Academy of Sciences (ÖAW). References 1. Oda, Y., Owa, T., Sato, T., Boucher, B., Daniels, S., Yamanaka, H., Shinohara, Y., Yokoi, A., Kuromitsu, J., and Nagasu, T. (2003) Quantitative chemical proteomics for identifying candidate drug targets, Anal Chem 75, 2159–2165. 2. Rix, U., and Superti-Furga, G. (2009) Target profiling of small molecules by chemical pro- teomics, Nat Chem Biol 5, 616–624. 3. Han, S.-Y., and Kim, Y.-A. (2004) Recent development of peptide coupling reagents in organic synthesis, Tetrahedron 60, 2447–2467. 4. Anderson, G. W., Zimmerman, J. E., and Callahan, F. M. (1963) N-Hydroxysuccinimide esters in peptide synthesis, J. Am. Chem. Soc. 85, 3039. 5. Neises, B., and Steglich, W. (1978) Simple method for the esterification of carboxylic acids, Angew. Chem. Int. Ed. Engl. 17, 522–524. 6. Kocienski, P. J. (2005) Protecting Groups, 3rd ed., Thieme, Stuttgart. 7. Rix, U., Hantschel, O., Durnberger, G., Remsing Rix, L. L., Planyavsky, M., Fernbach, N. V., Kaupe, I., Bennett, K. L., Valent, P., Colinge, J., Kocher, T., and Superti-Furga, G. (2007) Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel kinase and nonkinase tar- gets, Blood 110, 4055–4063. 8. Lombardo, L. J., Lee, F. Y., Chen, P., Norris, D., Barrish, J. C., Behnia, K., Castaneda, S., Cornelius, L. A., Das, J., Doweyko, A. M.,
  • 51. Another Random Scribd Document with Unrelated Content
  • 52. wines, wool, and other products are numerous, but unimportant. The iron ore mines (red and brown hematite) in the Somorrostro range and district are largely in the hands of English capitalists. These mines, which began to attract the attention of British iron masters about 1870, occur chiefly in the mountain limestone, and are worked in open quarries. Short railways and tramways have been made to San Nicolas on the Nervion; and a wire tramway has been constructed by the Galdames Mining Company, who possess a cliff of iron ore about a mile long and 280 feet high. The tramway carries the ore through a tunnel, 600 feet long, to the quay. The Landore Siamese Steel Company have important hematite mines connected with the river by a wire tramway, carrying baskets for loading. BILBAO—THE ORCONERO IRON ORE COMPANY’S WHARF IN LUCHANA. Bilbao is largely modern and wholly commercial, and its public buildings are not notable. But its thoroughfares are full of movement, and the shady arenal, in the old town—the focus of the life of the whole city—contains the principal hotels, the chief cafes, and the New Theatre. The land which this beautiful promenade now occupies was at one time very boggy, and swept by the tides. Now the two principal avenues are asphalted. The Church of San Nicolás de Bari, which faces it, is one of the city parish churches. It was built towards the end of the fifteenth century on the ruins of the sailors’ and fishermen’s little church. This church has suffered greatly on account of floods, especially during the year 1553. It was closed in 1740 as ruin threatened it. When it fell, the present one was begun in 1743. During the last war it was used as a provisioning station; and, after repairs, was opened for worship on the 21st of January, 1881.
  • 53. A GALICIAN. T In Northern Spain. HE great bulk of the Spanish people know as little of Galicia and the neighbouring Principality of the Asturias as the average Englishman knows of the Hebrides. Nor can they judge of the inhabitants of these provinces from the few individual Galicians who emigrate to Madrid any more than we in England can form an idea of Italians from the specimens who perambulate the London streets with a piano organ and a monkey. The Madrileño comes across a few Galicians in the capital engaged in menial services, and speaking a harsh, strange patois, which he finds some difficulty in understanding; but the Gallegan in exile is a very different person from the man you meet in his own land of rain and mist, where the scenery is exquisite, the hotels are famously bad, and devotion is the chief recreation of the community. At home these people are poor, but hardy; possessing little intelligence, but great capacity for work; knowing little comfort, but nursing a passionate attachment for the country of their birth. Many of the young women are remarkably handsome, but drudgery and hardship early tell their tale, and very few of them retain their good looks beyond the age of twenty. The country, for the most part, is poor to barrenness; the peasantry work day and night for mere subsistance; the cottages, which do duty for bedroom and nursery, stable, kitchen, rabbit hutch, pigsty and parlour, are damp and dirty, and destitute of beds or chimneys. The climate is rainy, the surface is mountainous, and the roads are generally bad. Small wonder is it that muleteers and commercial travellers constitute the principal visitors to Galicia—for those who have a soul above scenery, and an ambition beyond fishing, the country is practically without attraction. The single province of Oviedo, which constitutes the principality of the Asturias, harbours a people who have remained unconquered alike by Roman and Moor. There is protection, if not complete safety, in a country of mountain and valley, of damp and cold; and the Asturians have ever been able to spread themselves over the land and farm their straggling holdings in comparative security. They have cultivated maize for their staple food,
  • 54. A GALICIAN. A GALICIAN. poached the hills and rivers for game and fish, cultivated the art of dancing, and lived in terror of the evil eye from the most ancient times; and despite damp, hard fare, and harder toil, they have learnt REDONDELA (PROVINCE OF PONTEVEDRA)—GENERAL VIEW. the secret of longevity and the charm of a gracious civility of manner. Minerals in abundance are common to both Asturias and Galicia; and while the former is the richer in coal and iron, the latter has been worked for gold, silver, and tin from the time of the Roman occupation. It is on their mineral resources that these provinces will have to depend for their future prosperity.
  • 55. IN GALICIA. IN GALICIA. After the cities of the South— Barcelona, Toledo, Granada, or even modern Madrid—the Northern towns are small, shabby, and unimportant. Coruña, the chief seaport of Galicia, though interesting to Englishmen as being the landing place in Spain of John of Gaunt, and the harbour from which the invincible Armada sailed to conquer and Romanise Great Britain, is a place of only secondary importance. The city was founded by the Phœnicians; its name is probably derived from Columna, the Phœnician Pharos, or lighthouse; and its famous lighthouse, the Tower of Hercules, has had its counterpart from the earliest days. The Phœnicians, who made gain rather than discovery the aim of all their expeditions, were attracted to Galicia and to the province of Orense particularly by reason of its rich deposits of tin. Coruña in ancient days was the principal port of the North-west Coast, and the most westerly town in Europe. It is still the chief military station in Northern Spain, and ranks as a commercial city of the first importance. CORUÑA—GENERAL VIEW TAKEN FROM THE OLD TOWN. The hill-girt city of Santiago, though knowing nothing of commercial prestige, and having no part in the military system of the country, is to the traveller of far more interest than the capital of the province. For dead as it
  • 56. now appears to be, with the hand of death on its crooked, branching streets, and its crazy, deformed squares, which echo the pilgrims’ footfalls to the deaf ears of the dead, it was at one time the most celebrated religious centre in Spain—the goal of fanatics from every corner of Europe, the Mecca of countless thousands of theologians, and the tomb of one of the personal companions of Christ. Although the ancient glory of Santiago has departed, although PONTEVEDRA—GENERAL VIEW. its broad-flagged pavements are no longer thronged by the feet of the devout, and it has been much shorn of its former civil and religious dignities, the city is still the See of an Archbishop with a cathedral, two allegiate churches, and fifteen parishes. The cathedral is erected on the site of the chapel which was erected by Alonso II. to mark the spot where Theodomer, Bishop of Iria Flavia, is said to have discovered the body of St. James the Apostle; and the city, which sprang up around the memorial, bears the Spanish name for St. James the Elder. The original cathedral, which was finished in 879, consecrated in 899, and destroyed by the Moors in 997, was replaced by the present edifice in 1078. Whether one believes or not the tradition of the foundation of the cathedral—which, by the way, is no mere
  • 57. tradition in the mind of the Galician—one cannot but regard this mighty pile of stone with awe, and recognise in it the expression of an influence which was once felt throughout the Christian world. Even to-day it is one of the most frequented pilgrim-resorts in Europe. One passes through Pontevedra, a picturesque granite town, with arcaded streets and ancient houses bearing armorial shields, on the journey to Vigo. Here, as everywhere on the Galician coast line, the parish priest goes down to the shore one day in every year and blesses the sea; here also the oysters are excellent and abundant, and here the watchman’s night chant is heard in the streets. The call of the sereno, or watchman, who dates from the building of the ancient walls of Pontevedra, and the chapel of Alonso II. of Santiago, seems to catch the imagination of the traveller, and hurl him back into the mediæval ages, when life was a state that men fought to retain, and religion was a power for which they laid it down. The sereno, with his theatrical cloak wrapped about him, his axe-headed staff, his lantern, his majestic stalking walk, and his thrilling chant, “Ave Maria Purissima. Son las diez y sereno,” seemed to me impressive, unreal, almost fantastic. At ten o’clock he passed me in the deserted square, at eleven he was offering up his quavering invocation beneath my window. Galicia has little in common with the towns of the South—it retires to rest early in order to be up betimes. At Vigo a small fragment of the ancient sea walls yet remain, but the ruins that Lord Cobham made of the town in 1719 have been obliterated, and in place of the fortified port, which Drake visited in 1585 and 1589, we have a thriving, modernised town. Vigo is an important place of call for Mediterranean steamers, it is one of the chief centres of the cattle trade export to London, and the port of the mineral provinces of Pontevedra and Orense. The town of Orense, the capital of its province, is reached by the magnificent old bridge that spans the river Miño. Though now deprived of three of its arches, which were removed to give the road more width, and also of the ancient castle which defended the entrance, it continues to attract the attention of the traveller on account of its elegant and bold construction, its ample proportions and majestic appearance. Tradition says it is Roman, but many learned writers find nothing to confirm this assertion. It is quite likely that a bridge existed there previously; but the present one, it would appear, was built by order of Bishop Lorenzo during the first half of the thirteenth century, and has since undergone many alterations, including those
  • 58. to the largest arch, which is more than forty-three metres in width, and the reconstruction of which was completed about the middle of the fifteenth century. In the Roman days Orense was celebrated for its warm baths. These three springs, which are still in existence, flow copiously from fountains one above another, but the waters have lost their medicinal virtues—it is VIGO—VIEW FROM THE CASTLE. only a supposition that they ever possessed any—and are now used for domestic purposes. The present cathedral, which is an obvious imitation of the cathedral at Santiago, was raised in 1220. The cathedral, the warm springs, and the bridge over the Miño, comprise the three marvels of the city.
  • 59. GIJON—THE WHARF. Equally ancient, but in many ways more interesting, is the capital town of Lugo. It boasts a cathedral which shares with San Isidoro of León the immemorial right to have the consecrated Host always exposed; Roman walls in an excellent state of preservation that entirely surround the city, and an establishment of baths. The bath-house contains 200 beds; and the springs, which contain nitre and antimony, are good for cutaneous diseases and rheumatism. The river Miño, which is the glory not only of Lugo but of Galicia, rises in the mountains, some nineteen miles from the city. As the centre of a beautiful and variegated country, which affords good sport for the angler, and scenery of enchanting loveliness to attract the artist, Oriedo, the capital of the Astionas, has its charms; but the seaport of Gijon, with its tobacco manufactory, its railway workshops, its iron foundry, and glass and pottery works, is a much more thriving and important town. Gijon, like Santander, is a flourishing port; and both have gained immensely in importance of late years. While the latter, with its handsome modern houses, makes a more splendid show, its drainage and sanitary arrangements leave much to be desired, and the harbour at low water is sometimes most offensive. Both towns are of Roman origin, but Gijon is the most pleasantly situated on a projecting headland beneath the shelter of the hill of Santa Catalina, and the harbour is the safest on the North Coast. It exports apples and nuts in enormous quantities, coal, and iron, and jet; while its shores are much frequented by bathers during the summer months.
  • 60. SANTANDER—THE PORT. SANTANDER—GENERAL VIEW. It is currently believed, and I have no reason to doubt the accuracy of the statement, that if a visitor in any town in England stops the first native he meets and inquires as to the objects of interest that the place possesses, he will be referred immediately to the principal hostelry of the town. If you wander in London, and ask your way about, you will be directed right across the city by references to public-houses, which are the only landmarks that the Cockney ever dreams of studying. In Spain, cathedrals are as ubiquitous as inns are in England. You may be sure of finding comfortable accommodation for man and beast in most English towns, and in the Peninsula you can be quite as confident of “bringing up” against a cathedral —if nothing else. In León, the capital of the province of the same name, and in Salamanca, the second city in the province, we find the same state of things existing—the cathedral first and the rest nowhere. Yet these two cities boast of a noble history of ancient splendour and old-time greatness, and with this—and their cathedrals—they appear to be content. León, in the time
  • 61. LEÓN—THE CATHEDRAL. LEÓN—CLOISTER IN CATHEDRAL. of Augustus, was the headquarters of the legion that defended the plains from the Asturian marauders; and when the Romans withdrew, it continued as an independent city to withstand the continued attacks of the Goths until 586. The city yielded to the Moor, was rescued by Ordoño I., and retaken by the Arabs with every accompaniment of inhuman atrocity. Its defences were rebuilt by Alonso V. nearly 400 years later, its houses were repeopled, and it continued to be the capital of the Kings of León until the court was removed to Seville by Don Pedro. Its present miserable condition is a lamentable appendix to such a history. Its streets are mean, its shops are miserable, and its inns are worse. Nothing is left to it but its cathedral. This temple is truly an architectural wonder, combining the delicacy of the purest Gothic style with a solidity which has stood for centuries; the manner in which the problem of stability was solved is wonderful, the immense weights seeming to have no solid bases. The finest and most beautiful chiselled work is visible everywhere, and careful study is necessary in order to understand how the weight and strain of the arches were made to rest on their elegant buttresses. The origin of this magnificent temple is not quite clear, but many archæologists believe that it was founded in the time of King Ordoño II. It is of irregular form, but the cathedral or nave, transept, and presbytery are in the form of a perfect Latin cross.
  • 62. LEÓN—THE CATHEDRAL CHOIR STALLS. The windows are of colossal dimensions, and the ratablos and sculptures are notable. Among its many famous works the cloister must not be forgotten. It is an example of the transition style from ogive to renaissance, with large galleries, interesting groups of sculpture, and a beautiful door leading into the temple. LEÓN—VIEW TAKEN FROM THE CEMETERY. Among all the choral stalls treasured in Spanish churches those in the cathedral at León stand out prominently. Unfortunately, the names of the master who designed them, and of the artists who assisted him to carry that marvel of ogive art into effect, are not known; but it must have been executed during the last thirty years of the fifteenth century, for it is known that in 1468 the necessary bulls were obtained from his holiness through Archbishop Antonio de Veneris in order to arrange means for meeting the cost of the stalls, and in 1481 the work was still proceeding.
  • 63. SALAMANCA—GENERAL VIEW. Salamanca has a great name, a florid Gothic cathedral, and a square of handsome proportions and pleasant prospects. In other respects, it is quite without attractions. The streets are badly paved and dull, the climate is shrewd, and fuel, I was told, is scarce and expensive. Even the cathedral, though grand, is bare; and when one has visited the cathedral and lingered awhile in the pleasant garden of the Plaza Mayor—one of the largest and handsomest squares in Spain—and tested the accommodation of “La Comercio,” one can find little else to entrance one in the disappointing old city which was once a world-famed seat of learning. In the fifteenth century, when its university gave precedence to Oxford alone, it boasted of 10,000 students. In the following century its scholars had declined to one half that number, and to-day only some few hundred students are on its books. The sun of Salamanca commenced to set at a period of the world’s history that to all the rest of Europe was one of awakening and advancement. Decline and decay are writ large on the face of the city. From a distance its noble situation and fine buildings, built of beautiful creamy stone, gives the place an imposing and picturesque appearance. But though the shell of Salamanca remains, its spirit has departed. The ravages of the Romans, the Goths, the Moors, the Spaniards, and the ruin which the neighbourly French inflicted less than a hundred years ago, have left their cruel marks upon its historic walls. Salamanca is but a broken hulk spent by the storms that, from time to time, have devastated her. Her narrow, tortuous, ill-paved streets, which skirt its multitude of grandiose buildings, her squalor and poverty, her inferior art work, but even more the uncorrupted art of the grand old cathedral, all remind us of what Salamanca was, and turn our eyes backwards from what it is.
  • 64. SALAMANCA—VIEW OF THE COLLEGE FROM THE IRLANDESES. ZARAGOZA—“INDEPENDENCIA” PROMENADE.
  • 65. ZARAGOZA—PILAR CHURCH. One must approach Zaragoza with one’s mind full of memories of heroes, queens, poets, and bandits that have been associated with this once mighty city, and one’s heart filled with sympathy and respect for the old, proud Aragon that flourished, and was illustrious in history while the Englanders still decorated themselves with blue paint, and were domiciled in caves. For Zaragoza is not altogether a gay or an exhilarating city. Many of the streets have a gloomy aspect, and the old houses are high, dark, and repellant. But the city is not only important as the seat of a university, an Audiencia, an archbishop, the captain-general of Aragón, and other officials; it is also the junction of four railways, and its commercial progress has been steadily increasing of recent years. For Zaragoza is in reality two cities—the old part with ancient fortified houses, converted now into stables and wood stores, and the new part traversed by broad, well-paved, and excellently-lighted streets, and lined with modern buildings. Until the railway connected the city with Madrid and Barcelona, Zaragoza was as dead as Salamanca, and as dilapidated as León. But it has always held the advantage of those places in having two cathedrals to their one. The principal cathedral, that of La Seo, is a venerable Gothic pile occupying the site of a Moorish mosque, and its high arches have echoed many councils, and looked down on the solemn coronations of the kings of Aragon. More modern is the Cathedral El Pilar, so called from the identical pillar on which the Virgin descended from heaven. It was commenced on St. James’s Day, 1686, the work being designed and carried out by the famous Don Francisco Herrera, the architect. In the year 1753 King Ferdinand VI. instructed Ventura Rodriguex, the architect, to design and build a new church, as luxurious as possible, in which to instal the image without taking it out of its temple. This was done by erecting a small Corinthian temple under the magnificent cupola, which was ornamented with the richest marble and jasper that could be procured. On one of the altars of this temple, which is crowned with a magnificent silver canopy, reposes the venerated effigy, the jewels on which are of incalculable value.
  • 66. A FLEMISH DANCE. AT NUEVALOS. The Stone Monastery at Nuevalos, on the right bank of the river from which it takes its name, is one of the places most worthy of a visit in the province of Zaragoza, not only on account of the building itself, which is of great historical interest, having been built in 1195, but for the delicious picturesqueness of the place. Surrounded by rocks, winding amidst thick woods and dashing into deep abysses, this river runs its erratic course, imparting life to a landscape which is, according to the noted poet, Don Ramon Campoamor, “an improved dream of Virgil.” Among its many picturesque waterfalls, the one called “La Caprichosa” is perhaps the most beautiful. The dress of the Aragonese peasantry is peculiar and picturesque. The men, as a rule, wear no hats, but have instead a coloured handkerchief wound round the head, leaving the top bare. Their knee-breeches are slashed down the sides and tied by strings below the knee. The waistcoats are worn open. Round the waist they wind a wide sash, in the folds of which pipes, tobacco, money, and provisions are carried as safely as in a pocket. Their feet are shod with sandals, and they universally carry a blanket, which is thrown in a graceful manner over their shoulders.
  • 67. A Bull-fighting. BULL-FIGHT is underlined for an early visit in the note-book of every visitor to Spain. He goes prepared to be disgusted, and he comes away to denounce it as a revolting and demoralising exhibition. He even plumes himself upon his moral and human superiority over the Spaniard, because the spectacle proves too strong for his untutored stomach. The inference is as gratuitous as it is illogical. In point of fact, the effect of the spectacle upon the spectator is not so much a matter of sensibility as custom. The Spaniard grows up to the sport as our Elizabethan ancestors grew to bull-baiting—even as the present generation of Englishman grows to pugilism. To the Spaniard, the cruelty of the craft of tauromachy does not appeal; the spectacle inflames his blood, and stirs not a chord of compassion in his nature. Yet he can be intensely sympathetic, gentle, and tender- hearted; but these softer qualities of character are not touched by the sight of animal suffering. In the first place, the bull is his enemy by heredited tendency. He cannot forbear to hurl insulting epithets at him when he chances to pass him on a journey. He witnesses his end with the thrill of satisfaction which a soldier feels in the death of a treacherous and implacable foe. The Englishman cannot share, or even realise this sentiment —it would be strange if he could. His leading feeling is curiosity, and a nervous apprehensive tension which only magnifies the horror and repulsion of the sport. With the Spaniard it is entirely different. Long habit has familiarised him with the bloody details, and his experienced eyes follow each trick and turn of the contest with the enthusiasm of an athlete watching an athletic display. Every detail of skill and dexterity and nerve exhibited by the fighters, and every clever move made by the bull is greeted with critical applause. Cruelty there must be, but courage in a high degree is a factor in the contest—danger gives to the contest a dignity which is absent from pheasant shooting, and which formed no excuse for the vogue to which bear- baiting and cock-fighting once attained in this country.
  • 68. THE PROCESSION. It may be thought that I am trying to champion an institution which is regarded with aversion by all classes of English people, but such is not my intention. My object is to look at it from the Spanish point of view, and endeavour to see if there is not some plausible explanation of its popularity as a national amusement. But when all is said and done, there still exist two objections to the sport which cannot be explained away. The first is the almost inexplicable indifference which a Spanish audience shows for the torture that is inflicted upon the horses that take part in the corrida: the other is the attendance of the gentler sex. It must, however, be noted that a large proportion—certainly the majority of Spanish ladies—are opposed to the sport, and with the rest it is the manly courage and address of the performers that fascinates them. But the fact remains that women are seen in large numbers in the amphitheatre, as 300 years ago good Queen Bess was not ashamed to be a spectator at many an exhibition of bear-baiting. English sentiments in matters of sport have undergone a great change since the Elizabethan era, but Spain is notoriously the most conservative country in Europe. However, enough has been said of the theoretical side of bull-fighting; let us accompany the seething populace to the Plaza de Toros, and witness the sport for ourselves. The streets of Madrid are crowded with people who are all moving in the same direction. April to October is the regular bull-fighting season, but the Spaniard finds the lightest excuse a sufficient one for indulgence in his favourite pastime during the “close” season. And so, although it is February when I am in Madrid, I am not to forego an experience of a promising corrida.
  • 69. Although I have seen bull-fights in some of the best rings in Spain, including those of San Sebastian, Valencia, Barcelona, and Madrid, it is more particularly of my experiences at the latter place that I shall write. During the fashionable months, a boletin de Sombra, or “ticket for the shade,” is a luxury to be prized; but in February, in Madrid, we need all the warmth and glare that the sun can give us. The present Bull Ring, which was built at a cost of £80,000, and opened in 1874, seats 15,000 persons. It stands on a gentle elevation in a broad stretch of bare yellow land, where it raises its brick-coloured walls—the only land-mark in the barren, treeless, desolate expanse between the city and the solemn distant mountains. Around the various entrances countless human beings cluster like bees, and the Plaza is alive with men and horses, mules with tinkling bells, soldiers, police, picadors, and fruit-sellers. What strikes one most curiously about this concourse of human beings, both outside the bull-ring and within the huge amphitheatre, which rises tier above tier from the brown sand till it is almost lost in the vast expanse of blue above, is its single-mindedness, its patience, and the entire absence of horseplay. To a Spaniard this is not curious, but to the English spectator some familiar characteristic of a crowd appears to be absent. ENTRANCE OF THE BULL. Punctuality is not a strong trait in the Spanish character, but punctuality will be observed to-day. At the hour and the minute appointed, the President enters his palco, the signal is given, and the proceedings commence. The procession, headed by two caballeros, habited in black velvet, moves slowly across the ring to the front of the President’s seat. The two espadas in yellow and violet, and gold and green costumes respectively, follow the caballeros. After them come half-a-dozen stoutly-protected picadores, then eight
  • 70. banderilleros, gay with a profusion of silk sashes, short breeches, and variously-coloured hose, and the rear is brought up by a posse of attendants, leading the mules, all bedecked in plumes and rich trappings, which are to drag off the carcases from the arena. The entrance of the glittering cavalcade is announced by a trumpet sound, and the President tosses the key of the toril into the ring. To the “new chum,” all this preliminary detail, commonplace and “circusy” as it is, is sufficient to strain the nerves, and expectancy changes to apprehension. The creak emitted by the opening of the heavy door of the toril intensifies the feeling. The clutch of curiosity with which the entire concourse awaits the entrance of the first bull is contagious. Instinctively one strains forward and catches one’s breath. Toro does not keep us long in suspense. There is a momentary lull, and then the bull dashes from his dark cell into the glint of the Spring sunshine. The novelty of the environment staggers him for a moment. He hesitates in the centre of the ring, and looks wildly around him. The arena is empty, with the exception of three picadores, who sit rigidly in a row on their sorry hacks, waiting for the bull to recognise their presence. Our first victim is a doughty warrior. He is as ignorant as the blindfold knackers—that would be dear at a pound a leg—of the fate in store for him. He may make a brave fight, kill horses, upset men, and leap the barriers with a heroic rush, but in twenty minutes his corpse will be coupled up to the mules, and fresh sand will be strewn on the red trail that will mark his last passage across the arena. The inevitableness of the outcome of the encounter, so far as the principal actor is concerned, is the least pleasing feature of the sport. The fox and the stag are
  • 71. ANTONI O FUENTE S. LUIS MAZZANTINI AND CUADRILLA. U E R R I T A . B a n d i l l e r o . given a gambling chance, the grouse is not without hope, and the gladiator of the cock-pit may live to fight another day, but the bull is a doomed animal. Happily he is not capable of calculating the uselessness of his efforts. The horses stand but little better chance, and the picadores, despite their iron and leather greaves and spears, are paid to take risks. The art of the picador is displayed in the skill with which he avoids the charge of the bull, and turns him on to the next picador, who, in turn, will pass him on to the third. In this instance the manœuvre does not come off. The bull’s rush is met by the first picador with the point, but the horse he strides is too ancient to obey with sufficient celerity the rider’s injunction to swerve, and horse and man are rolled over with the force of the impact. The
  • 72. wretched equine is lacerated on his opposing flank, but the spearman appears to be uninjured, and before the bull has completed his circuit of the ring, the horse is on his feet again, and the picador is waiting for the next attack. The toreros, with their red capa, are immediately on the spot to draw the bull from his victim, but the bull is too eager to waste time on a fallen foe. The second and third horseman avoid his rush; and the bull, smarting from spear thrusts, and confused by the cheers, is inclined, in racing parlance, to “turn it up.” The first horse who crosses the line of sight is caught on the brute’s horns, and is so deeply impaled that the bull has to swerve at right angles to rid himself of his enemy. The second horse is impaled before the combatant can plant his spear in the bull’s neck. Steed and rider are lurched in the air, and fall heavily to the ground, and the momentary victor lowers his head again to the prostrate man, and rolls him over and over. Toreros hasten to the spot to get him away, the people rise in their places, ladies lift their fans and avert their faces, while the air is filled with the usual murmur of lamentation which accompanies an accident. Both the other picadores are unhorsed before the President gives the signal for them to retire. Act one of this most realistic of sporting melodramas is over. The banderilleros now come forward. They are costumed like Figaro, in the opera of “Il Barbiere de Sevilla,” and their hair is tied into a knot behind. To the English spectator, this part of the performance is the most fascinating and least abhorrent of the entire piece. The banderillero inflicts no more pain on the bull than the humane angler deals out to the wily trout, and the agility and daring with which he addresses himself to his task is superb. His aim is to plant small barbed darts, or banderillas, on each side of the neck of the bull. The chulos, or apprentices, here open the ball by tantalising the animal, and working him up to a proper pitch of fury. Then the banderilleros circle round him, and one, standing full in his line of flight, “defies” him with the arms raised high over his head. If the bull stops, as he is doing now, the man walks composedly towards him. Then the bull lowers his head and makes his rush, and the athlete, swerving nimbly to one side, pins in his banderillas simultaneously. Again and again the maddened animal, frantic more from impotence than pain, makes his rushes from one tormentor to another. At each rush he receives further instalments of his hated decorations. Then one man bungles. He loses his nerve, or, failing to time the animal’s charge, shirks the onslaught. A howl of execration greets the exhibition, and the unfortunate baiter is tempted to more rash efforts. He seats himself in a
  • 73. chair, and waits with suicidal calmness the rush of the bull. Just as the animal’s horns are thrust beneath him he jumps lightly up, manipulating his darts with miraculous precision, while the chair is tossed high in the air. Thunders of applause greet this venturesome feat, and the other banderilleros, warmed to their work by the plaudits of the public, vie with one another in deeds of coolness and “derring do.” One waits, alert but motionless, for the attacks of the charging bull, and as the galloping brute lowers his head to toss him, places his foot between the terrible horns, and is lifted clear over his onrushing enemy. Another, seizing hold of the lashing tail, swings himself along the bull’s side, and plants himself for one thrilling moment right between the horns. THE PICADOR. I once saw a banderillero, in response to the jeers of the crowd, take the darts, which are about two feet long, break them across his knee, and plant the stumpy weapons, with unerring precision, on each side of the neck of the bull. These feats appear to be fraught with infinite danger, and the agility with which the performers acquit themselves cannot be witnessed without a tremour of amazement and admiration. Several times the venturesome chulos escape death as by a miracle: they sometimes seem so close to their end when they vault over the barriers to avoid the pursuing bull, that they appear to be helped over the fence by the bull’s horns. One bull exhibits at this stage of the proceedings an emphatic disinclination to continue the fight. He paws the ground when the darts are driven home, but makes no show of retaliation, and the hoots and opprobrious epithets that are hurled at him by the populace fail to inspire him to renewed efforts. Then the banderillas de
  • 74. fuego are called for. These are arrows, provided with fire crackers, which explode the moment they are affixed in the neck. In a moment the spectacle, which had worked me up to a high pitch of excitement, becomes intensely distasteful. The tortured animal, driven mad with fright and pain, bounds across the ring in a series of leaps like a kid. The people scream with delight, and I mentally wonder what kind of “steadier” the Spaniard resorts to when his stomachic nerve is affected by a detail of the exhibition. The firework display had not lasted long when the last trumpet sounded, and the espada walks forward to a storm of rapturous applause. The finale of the spectacle is approaching. The executioner comes alone: the bull, who has hitherto been tormented by a crowd of enemies, is now able to concentrate his whole attention on one object. Toro has become exhausted with his previous exertions, and he moves without his old dash. The espada studies his foe carefully, to judge the temper of the animal with which he has to deal. With his left hand he waves the muleta—the red cloak —to lure the beast into a few characteristic rushes and disclose his disposition. If he is a dull, heavy bull, he will be despatched with the beautiful half-volley; but if he proves himself a sly, dangerous customer, that is cunning enough to run at the man, instead of at the muleta, a less picturesque, but safer thrust must be employed. But our bull is neither sly nor leaden. He has recovered from his fright, and is quick to seize his opportunity to make a final effort before the stinging banderilleros return to distract him. Once or twice he thrusts his horns into the unresisting cloak, then gathers himself together for a final rush. The swordsman raises the point of his glimmering Toledo blade; while every nerve of his sinuous, graceful body quivers with the absolute constraint and concentrated effort that hold him. The duellists are both of the same mind. The espada has summed up his antagonist—he is levantados, the bold bull, a fit subject for la suerte de frente. The bull’s next rush is his last. The fencer receives the charge on his sword, which enters just between the left shoulder and the blade. The bull staggers, lurches heavily on to his knees, and rolls over, at the feet of his conqueror, vomiting blood. The assembled multitude rend the air with their cheers, the men yell applause, and every face is distorted with excitement and enthusiasm. The only indifferent person in the building is the espada. With a graceful and unassertive turn of his wrist, he waves the sword over his fallen foe, wipes the hot blood from the blade, and turning on his heel, approaches the
  • 75. President’s box, and bows with admirable sang-froid. The team of jingling mules enter, and the dead bull is carried off at a rapid gallop. The espada walks composedly away, without another glance at the result of his handiwork. The superb imperturbability of these espadas always fills me with admiration. They accept the plaudits of the spectators with the same unconcern with which they hear the execrations that fill the air if they do not at the first attempt inflict the coup de grace. During the first corrida I attended, an espada failed to aim at the precise spot, and the bull tore up the sand in agony. The populace insulted the swordsman with jeers and howlings, but he remained perfectly cool and collected, and nerved himself with as much composure to his second and successful thrust as if he had been practising with a sack of potatoes in an empty arena. When I had been witness to the death of two bulls, I remarked to my Spanish friend that I had seen as much as I desired, and was quite ready to quit the spot. But my companion was a friend of long standing: he could be firm without seeming discourteous. “No! no!” he said, “you kept me in the theatre last night until ‘Don Juan’ was played to the bitter end: you shall remain to-day to reward me for my exemplary patience and respect for your wishes.” I saw five other bulls done to death during the afternoon. AT CLOSE QUARTERS. Although not to be compared with an ordinary corrida as a display of skill, and capacity, and artistic finish, a Royal bull-fight, such as Madrid saw on the occasion of the coronation of King Alfonso XIII., is more interesting as being a revival of the sport as it was originally practised. Bull-fighting to- day is a purely professional business, but in the knightly days of ancient Spain it was employed as a means to teach the chivalrous youth the use of
  • 76. arms. In those days, mounted caballeros encountered the bulls in the ring with lances alone—a more dangerous pastime than is bull-fighting in its modern sufficiently hazardous form. Then the combatants were mounted on good horses, and their business was to save them and turn the bull, to kill the bull if possible, but, at the risk of their own lives, to protect their steeds from injury. It is recorded that in one Fiesta de Toros at the beginning of the sixteenth century, no less than ten young knights lost their lives. The corrida, Real con Caballeros en plaza—a Royal bull-fight with gentlemen in the arena—on the olden lines, that was held on May 21st, 1902, in Madrid, was fought by young officers and scions of noble families, who were attired in the gorgeous costumes of Spanish knights of the reign of Philip IV., and attended by their pages and grooms wearing the dress of the same period, and displaying the colours of the noble house which they served. On that occasion, the Paseo de las Cuadrillas, or preliminary procession of the bull- fighters across the arena to the strains of military music, was a most imposing sight. The Padrinos, the grandees who acted as supporters or godfathers of the knights, accompanied the fighters, followed by their mediævally-clad retinues, to the foot of the Royal box, and presented them to the King. The spectacle was strikingly brilliant, but the display was not to be compared with a professional bout. The horses of the cavaliers had evidently not been sufficiently trained for their work, and the best riding in the world could not bring them off scathless. Let me condense an account of the scene to convey an impression of what the present-day bull-fight has been derived from. When the procession had withdrawn, leaving only the chulos and the gallant caballeros in the arena, the door of the toril swung on its heavy hinges, and a splendid specimen of a bull, dungeoned for several hours previously in utter darkness, darted into the light of day, tearing up the ground with its hoofs, and ploughing the air with its horns. Suddenly, a horseman and his prancing steed vaulted into the centre of the ring—the charger, with flowing mane, full-veined ears and shapely head slanted forward—to meet the onrush of the goaded bull. The second picador seeing the bull worried and dazed by the tantalising assistants, scudded past on a swift, white racer, sitting gracefully in his saddle, and then turning deftly as he passed the great brute, plunged his lance into his neck, and whirled aside to avoid possible pursuit. But by sheer accident, the bleeding steer dashed
  • 77. off in the same direction, caught the horse in the hindquarters, raising it on its forelegs and endangering the equilibrium of the rider. Before the scampering bull had time to recover from the compact, the second caballero, dashing up, had planted his lance deep into its neck. The white horse, stung with pain, made a wild rush, but was brought to hand by splendid horsemanship, and his rider urged him along, to inflict another wound in the animal’s head. Then two toreros advanced, beguiling and wearying the bull. By the time the bull had received the fifth lance in his neck, and the white steed had been twice wounded, the edge was taken off the keen thirst for violent emotions, and another torero unfolded his red capa, waved it to and fro until the bull swooped down upon him, and a moment later he was sprawling in the sand seemingly gored by the infuriated animal. The next minute the wounded steer tottered, dropped on its forelegs, and turned over on the sand, and a knife put a speedy end to its sufferings. The second bull, a black massive creature, appeared listless and faint, and made little effort to defend itself. It made one successful attack on the white charger; and, then, at the signal from the King, an amateur espada stepped forward. The attempt was a miserable failure. The young swordsman dedicated, in a few well-chosen words, the death of the bull to his sovereign, and after a dozen passes with the red capa, plunged the gleaming blade of Toledo steel into the animal’s neck, but so ineffectually that a storm of hisses resounded through the ring. The second attempt was still more awkward, the sword entering but a few inches. The sword was pulled out, and another effort, made amid groans and hisses, proved equally unsuccessful. A TURN WITH HIS BACK TO THE BULL. Although the madness had died out of the expiring brute’s eyes, and his forelegs were bending under him, the inexperienced torero seemed unable to
  • 78. put him out of pain. However, he grasped the short, sharp knife, and unsteadily taking aim, plunged it into the neck. Another failure. Yells, groans, shrieks, whistling, and hissing marked the anger of the crowd. The espada may be a paid professional, or the greatest noble in Spain, but in the ring he is judged by the rules of the ring, and his bungling is recognised with the most poignant scorn to which failure could be subjected. He again grasped the sword; and, spurred by the vitriolic exclamations of the public, sheathed it in the bull’s neck. The animal stood still and tottered, his forelegs bent, his head sank upon the moist, red sand, his hind feet quivered, and a flourish of trumpets announced that life was extinct. It is curious to find, in talking with learned enthusiasts on the relative merits of the bull-fighters, what diversity of opinion exists; but all parties are agreed upon the unrivalled skill and daring of the mighty Frascuelo. In his day, for death’s whistle summoned him from the arena in the height of his fame, Frascuelo was regarded as the greatest matador that Spain had ever seen; and Spaniards, in debating the subject of the bull-ring, never indulge the hope that his equal will ever arise to shed a new glory on the National sport. Frascuelo is dead, and his famous rival, Guerra, or Guerrita—to give him his professional name—has long since cut off his coleta, and lives in well-earned retirement at Córdova. But the school of fighters, who claim Frascuelo as their master—the fearless, dare-devil toreros, who scorn to concede a yard of ground to the bull, and do all their fighting at close quarters—is widely popular; and if their terribly dangerous methods are attended by frequent casualties, the intoxicating applause that rewards the accomplishment of a brilliant coup is, apparently, ample compensation for the risks that it entails. But the wildest appreciation of a successful feat does not exempt the most popular performer from the furious condemnation of the multitude when his scheme miscarries. The allowances made by a Spanish audience at the ring-side are of the most grudging nature. I once travelled from Barcelona to Madrid in the company of Bombita-Chico—the boy Bombita—who, although he was barely recovered from an unfortunate encounter with a tricky bull eight days before, was on his way to take part in a grand corrida that was to be held in the capital. He was—as his name denotes—no more than a lad, with large, strong hands that sparkled with jewels, while a formidable anchor about five inches long, set with magnificent diamonds, dangled from his watch-chain. I saw him again in the arena a few days later. He seemed nervous, and was, it appeared to me, a
  • 79. little perturbed by the demonstration that welcomed his reappearance in the ring after his accident. Ill fortune allotted him a troublesome animal, and his kill, while creditable enough to untutored eyes, lacked the grace and finish that the critical spectator requires. Bombita was their own Boy of Madrid, and because of his recent misfortune they forgave him, but they did not cheer him; and the lad walked out of the arena amid a silence that could be felt. FIXING THE BANDERILLAS. Mazantini, now grown old and heavy, was in his day an undoubtedly fine matador. There are some that still regard him as the head of his profession. But the majority, remembering what he was, regret that he has not gone into honourable retirement. But Mazantini cannot tear himself away from the fascination of the arena, although his appearances grow less frequent every year. Conejito, who was wounded in Barcelona in the spring of 1903, is generally regarded as the most accomplished matador now before the public; but Fuentes is, par excellence, the best all-round man. For, with the exception of the picador business, Fuentes plays every part in the piece. Other espadas have their assistants, who play the bull with their capas, and stand by while the banderilleros ply their infuriating darts. It is only when the bull has been prepared for the slaughter by the other performers that the matador comes forward to put the finishing touch to the grim tragedy. Fuentes, on the other hand, on special occasions—of which the corrida which I attended in Madrid was one—keeps his assistants entirely in the background; he takes the stage when the picadores leave it, and keeps it to the end. So close does he keep to the bull, that during the corrida in Madrid, of which I am writing, he seldom allowed the animal to be a dart’s length away from him. On one occasion his capa got caught so tightly on the bull’s
  • 80. horns that he tore it in jerking it away; and at another time the bull stopped dead, with his forefeet on the hated sash. As a banderillero, Fuentes is without equal in Spain. He frequently works with darts that have previously been broken short, and he uses them sparingly. Yet the encounter between the banderillero and the bull when Fuentes is on the scene is the most thrilling part of the whole performance. It is a contest between human intellect and brute intelligence—a duel between mind and matter. Fuentes does not avoid the bull, but by exerting some magnetic power he repulses the animal and compels it to halt. When the bull charges, in response to his “defiance,” he waits with the banderillas suspended above his head until the animal is within a few yards of him. Then he deliberately, but without haste, lowers one arm until the arrow is on a level with the brute’s eyes. The bull wavers in his onslaught, slows up, and stops dead within a foot or two of the point. Sometimes Fuentes walks backwards, while the bull glares at him with stupefied impotence, until he escapes the eyes that THE MATADOR. hold him, and gallops away. Again and again the banderillero taunts his enemy to attack him, only to arrest his charge and force him to turn from his deadly purpose by the irresistible power of his superior mentality. The crowd follows this superb exhibition with breathless interest, and in a silence that is more eloquent of admiration than the wildest cheers would be. But the end is nearly reached. Fuentes grasps his stumpy darts and advances against his bewildered antagonist, who waits his approach with sulky indifference. The man’s arms are flung up with a gesture of exasperating defiance, and when the bull makes his final rush, his opponent, instead of stopping him, steps lithely on one side, and the brute thunders past him with the two galling arrows firmly implanted in his huge neck. Fuentes has already moved to the
  • 81. side of the ring. The bull turns and charges back at him. The banderillero glides gracefully over the sand, but his pace is not equal to that of his infuriated pursuer. The distance between them decreases rapidly; in half-a- dozen yards he will be upon him. Fuentes glances over his shoulder and, without changing his pace, doffs his cap and flings it in the bull’s face. This stratagem only arrests the rush of the brute for a moment, but it gives the man time to reach the barrier, where he receives his muleta and sword from an attendant and returns to complete his task. All the kings of the bull-ring have their own particular feats or strokes, which the Spaniards appreciate as Englishmen revel in Ranjitsinhji’s acrobatic hitting, or Morny Cannon’s inimitable “finishes.” Bombita-Chico’s speciality in playing his bull is to kneel in the arena and allow the animal to charge through the capa which is held within three feet of the ground. The nerve required for this feat fires the audience with enthusiastic approval. The tale is told of a torero, whose name I have forgotten, who gained distinction by his exceptional skill in facing the bull with the long vaulting pole, known as the salto de la garrocha. With this instrument he would goad the bull on to the attack. When the brute was in full gallop he would, timing his movements to the instant, run a few yards to meet him, and swing himself high into the air at the end of his pole. The oncoming bull would charge the pole, the grounded end would be tossed upwards, and the torero would drop lightly to the ground and make good his escape. On one occasion the man performed his risky “turn” at a moment when the attention of a royal lady was attracted from the arena, and she sent an attendant to the expert to command him to repeat it. In vain the poor fellow protested that it was impossible for him to accomplish the same feat again with the same bull. The lady’s desire had been expressed. “But it is more than my life is worth,” argued the athlete. “It is the lady’s wish,” responded the attendant. The torero bowed, and “I dedicate my life to Her Royal Highness,” he said. The attempt fell out as he foretold. The bull charged and stopped dead. The man vaulted aloft, his body described a half circle, and fell—on the horns of the bull. He was dead before the attendants could entice the animal from his victim.
  • 82. THE FINAL STROKE. Lagartijo, Lagartijillo, Mazantini, and Montes all have their distinguishing methods of attacking and despatching the bull, but none of these are capable of the feat by which Guerrita was wont to throw the bull- ring into transports of deafening enthusiasm. In the ordinary way, the espada having taken the measure of his adversary, receives him standing sideways, and having thrust his sword at arm’s length between the left shoulder and the blade, leaps aside as the bull blunders forward on to his knees and falls to the earth. But Guerrita advanced his left arm across his body and waved his muleta under his right uplifted arm. When the bull lowered his head at the charge he passed the sword over the animal’s horns and plunged the blade into the vital spot behind the shoulder. In other words, he stopped the brute and killed him while his head was under his arm; and so closely were the duellists locked in that last embrace, that Guerrita’s side was frequently scratched by the bull’s horns. One may lecture, write, and preach against the barbarity of bull-fighting; but so long as Spain can breed men of such amazing nerve, and skill, and dexterity that they can successfully defy death and mutilation to provide their countrymen with such lurid sport, so long will bull-fighting continue to flourish in Spain.
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