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Spectral techniques in proteomics / editor, Daniel S. Sem.
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1. Proteins--Spectra. 2. Proteomics--Methodology. 3. Mass spectrometry. I.
Sem, Daniel S.
[DNLM: 1. Proteomics--methods. 2. Mass Spectrometry--methods. 3.
Spectrum Analysis--methods. QU 58.5 S741 2007]
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![xi
Preface
A significant challenge in presenting an overview of Spectral Techniques in Proteomics
is in defining the scope of the topic. Proteomics means different things to different
people; for years, the dominant technique employed was 2D gel electrophoresis,
followed by mass spectrometry (MS). While many exciting MS applications are
presented (e.g., matrix-assisted laser desorption/ionization [MALDI], electrospray
ionization [ESI], tandem MS, liquid chromatography [LC]-MS, surface-enhanced
laser desorption/ionization [SELDI], isotope-coded affinity tag [ICAT]), a comprehen-
sive survey of MS methods and applications in proteomics is certainly beyond the
scope of this book. Since Spectral Techniques in Proteomics is intended for a broad
audience of protein biochemists and biophysicists, topics such as structural proteomics
and chemical proteomics will also be covered, along with fluorescence/array-based
screening, SPR (surface plasmon resonance), and other “lab-on-a-chip” technologies.
Furthermore, a disproportionate amount of time will be spent on some less
established spectroscopic methods in proteomics, with forward-looking speculation
on future applications. The intention of this book is therefore to facilitate the inno-
vation, development, and application of new spectroscopic methods in proteomics
while giving a modest overview of existing and proven techniques. To this end, a
broader view of proteomics is taken in order to include studies that go beyond the
usual scope of 2D gels and MS, attempting to address function and mechanism at
the level of protein–ligand interactions. After all, this is the realm in which protein
spectroscopists have always excelled and felt most at home.
Proteomics is defined broadly as the “systems-based” study of proteins in the
organelles, cells, tissues, or organs of an organism. It is the study of the protein
complement of the genome in time and space. In practice, this definition sometimes
limits proteomics to the study of proteins in 2D gels, since this is one of the few
contexts in which so many proteins can be studied at once. It is also possible to simplify
a proteome into a more manageable subset (a subproteome) by focusing on a smaller
number of proteins related in a systems-based manner. Such systems of interrelated
proteins can include: (1) regulatory cascades connected via protein–protein inter-
actions; (2) metabolic pathways; (3) proteins with related modifications (acylation,
phosphorylation, glycosylation, methylation, etc.); and (4) any collection of proteins
associated with a biological effect, such as uncontrolled cell growth (cancer), an
immune response, or drug metabolism. This approach to simplifying a proteome
into systems-related subproteomes is described in chapter 1.
Thus, for purposes of this book, proteomic studies are extended to include the
parallel study of subsets of related proteins, some of which were described previously.
Such subsets might also include proteins that comprise a unique basis set of protein
folds in an organism’s proteome, as currently defines the scope of most structural
proteomic initiatives. Another systems-related subset could comprise proteins with
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![3
1 The Systems-Based
Approach to
Proteomics and
Chemical Proteomics
Daniel S. Sem
CONTENTS
1.1 Introduction...................................................................................................... 3
1.2 Complexity and Dynamic Range Challenges.................................................. 4
1.3 The Systems-Based Approach ......................................................................... 4
1.3.1 Systems-Based Relationships .............................................................. 4
1.3.2 Subproteomes....................................................................................... 6
1.3.3 Chemical Proteomics ........................................................................... 7
1.3.4 Applications ......................................................................................... 8
1.4 Summary .......................................................................................................... 9
1.5 Future Prospects............................................................................................. 10
References................................................................................................................ 11
1.1 INTRODUCTION
Simply stated, proteomics is the study of the protein complement of a genome using
the tools of protein biochemistry on a proteome-wide scale. It is devoted to monitoring
changes in expression levels or post-translational modifications of all the proteins in
an organism, organ, cell, or organelle as a function of time or biological state (e.g.,
diseased vs. healthy). Ideally, it should also address protein structure–function in
terms of interactions with substrates, drugs, inhibitors, lipids, DNA, or other proteins.
It is possible to infer some information about protein expression levels based on
changes in mRNA detected using microarray technology—an elegant coupling of
microfluidics, “lab-on-a-chip,” and detection (usually fluorescence-based) technolo-
gies. But, mRNA levels are not always correlated well with protein levels, and they
reveal nothing about post-translational modification or protein interactions. As such,
the field of proteomics serves an essential function, despite the additional technical
challenges involved in analyzing proteins in comparison with polynucleotides [1–4].
Most significant is the challenge of dealing with sample complexity and dynamic range.
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![4 Spectral Techniques in Proteomics
1.2 COMPLEXITY AND DYNAMIC RANGE CHALLENGES
The human genome comprises over 30,000 genes, which encode many more proteins;
many are variants due to alternative splicing and PTMs (post-translational modifica-
tions). Over 400 PTMs are known to date, so there is tremendous complexity in the
proteome as it is expressed in a given cell type. While it is a challenge to resolve the
thousands of proteins expressed in a proteome, it is an even greater challenge because
these proteins may be present at very different concentrations, ranging over six to
nine orders of magnitude depending on the cell type. For example, serum contains
albumin as the most abundant protein (at ~40 mg/mL and ~50% of blood protein),
while other proteins of interest, such as interleukin-6, are present at <5 pg/mL [5].
The need to quantify so many proteins over such a wide concentration range is one
of the greatest challenges in proteomics. Other challenges include the need to assess
interactions between the proteins and their various ligands that define biological
networks or systems. Furthermore, at least in some cases, these interactions should
also be measured within a cell (chapter 15) to ensure their biological relevance in the
context of potential accessory proteins or cofactors, as well as under physiologically
relevant conditions (water activity, pH, ionic strength, lipids, etc.).
1.3 THE SYSTEMS-BASED APPROACH
1.3.1 SYSTEMS-BASED RELATIONSHIPS
Proteomics is considered a subdiscipline of systems biology. So what is systems
biology? Weston and Hood define it as “the analysis of the relationships between
elements in a system in response to genetic or environmental perturbations, with the
goal of understanding the system or the emergent properties of the system” [6].
“System” is a broad term borrowed from other fields (e.g., engineering), but in a
biological context the word usually refers to organelles, cells, organs, or organisms.
Such a definition is therefore based largely on the physical location of the proteins
studied, their network of interactions, and their collective role in defining a bio-
logical entity (such as a mitochondrion or a liver cell) or function (such as the
immune response).
It is also possible to define systems at the level of molecules based on the network
of interactions that occur between them, usually within the context of a single cell
or organelle. This typically involves the measurement of all pairs of protein–protein
interactions that can occur, using techniques such as the yeast two-hybrid system.
In this manner it is possible to establish networks of proteins that participate in
interactions with each other. These pairs of interactions, summed across a whole
proteome, comprise a protein interaction map such as that shown in figure 1.1 and
discussed in chapter 14 by Rich and Myszka.
In a broad sense, systems or networks of interacting proteins can be defined
based on the presence of interactions between [7]:
(a) Two proteins directly, as in a regulatory cascade when a protein kinase
phosphorylates another protein
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![The Systems-Based Approach to Proteomics and Chemical Proteomics 7
Subproteomes categorized based on physical location in the cell are the easiest
to define since separation techniques permit isolation of organelles [8]. The most
general breakdown of cellular location is into the nucleus, cytoplasm, or membrane/
extracellular region. A more detailed breakdown would include the 14 categories of
subcellular locations and organelles defined in figure 1.1 [9]. Subproteomes defined
based on protein–ligand interactions are presented at greater length later in this book
(part IV) and fall largely within the scope of chemical proteomics, described in the
next section.
1.3.3 CHEMICAL PROTEOMICS
Chemical proteomics is a branch of proteomics [7,10–14] with a focus on directly
detected protein–ligand interactions measured across a systems-related group of
proteins. It is therefore a mechanistic complement to chemical genetics. In chemical
genetics, the observable is a phenotypic change induced by a chemical knockout
[15], but without any direct characterization of protein–ligand interactions. Bogyo
defined chemical proteomics as being focused on the structure, function, and role
of proteins in different biological systems using chemical probes [10]. Thus, the
field relies heavily on chemical probes to define systems-related proteins. These
probes can be activity based (covalently labeling all proteins that share a common
electrophile or nucleophile), as illustrated in the left side of figure 1.3.
An example might be labeling of all thiol groups, as done with the ICAT (isotope-
coded affinity tag) technology pioneered byAebersold [16] and presented in chapter 13.
FIGURE 1.3 Chemical proteomic studies. Such studies employ probes to “profile” proteins.
This can be done using: (a) activity-based profiling, with probes that covalently label proteins
(left arrow); or (b) affinity-based profiling, with probes that noncovalently bind to proteins
(right panel). (Adapted from Sem, D.S., Expert Rev. Proteomics, 1, 165–178, 2004.)
–OH
–S–
Proteome or Sub-proteome
Proteins related by:
(a) Binding site
shape
or
(b) Binding site
nucleophile
or electrophile
–OH –OH
–OH
–S–
–OH
–OH
–S
–S
–S–
–S–
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![8 Spectral Techniques in Proteomics
Detection of probes can also take place by using fluorescence, as developed by
Bogyo and others [10,17]. Probes can also be affinity based (right side of figure 1.3),
where they bind noncovalently to families of proteins [7,12,18] with related binding
sites (so-called pharmaco families [14,19,20]). Many subproteomes are isolated and
characterized based on common binding sites, so such classification is extremely
important for the fields of proteomics and chemical proteomics. It is also important
in drug design, where binding site similarities can determine how specific a drug is
for its intended protein target relative to antitargets, which include drug-metabolizing
enzymes as well as proteins that produce undesired side effects. The science of
classifying protein binding sites based on ligand-binding preferences is a growing
field that has evolved independently of proteomics. It is discussed in detail in chapter 2
by Villar et al.
1.3.4 APPLICATIONS
Most proteomic studies now employ mass spectral detection, usually of proteins
extracted from 2D gels (chapter 4) or fluorescence studies of microarrays (chapter 10
and fig. 1.4). An alternative to matrix-assisted laser desorption/ionization (MALDI)
analysis of extracted proteins is to use in-line purification of proteins by capillary
electrophoresis (CE), followed by electrospray ionization-mass spectrometry (ESI-MS)
(chapter 4). Although these methods provide good resolution, to help address the
complexity problem in proteomics, they usually also require further simplification
of proteome samples. The systems-based approaches described earlier for simplify-
ing proteomes are therefore crucial in most proteome studies. In particular, it is
FIGURE 1.4 Various subproteomes quantified using fluorescence detection of microarrays.
Different ways of capturing subproteomes are shown using antibodies, antigens, ligands, and
other affinity–capture arrays. Discussed further in chapter 10.
Antibody array Antigen array
Micro &
microarrays
Fluorescence
detection
Binding
Labeling
Antibody Ligand
Peptide
Protein
Phage displayed array Total protein array
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![The Systems-Based Approach to Proteomics and Chemical Proteomics 9
routine to analyze subproteomes rather than entire proteomes. This book presents
studies of many different subproteomes, including:
• immunoproteomes (chapters 9, 10)
• glycoproteomes (chapters 6, 13)
• phosphoproteomes (chapter 13)
• transcriptional regulatory pathways (chapter 11)
• ATP-binding proteins/protein kinases (chapter 18)
• the metabonomes (chapter 16)
Many of these studies require some technique to first purify the desired subpro-
teome, usually based on an affinity purification step before MS analysis. As an
alternative, surface-enhanced laser desorption/ionization-mass spectrometry
(SELDI-MS; chapter 7) employs affinity purification on the chip itself, which is
coated with an appropriate ligand that captures the desired subproteome (fig. 1.5).
Related affinity-capture techniques are also possible using microarrays (fig. 1.4).
Finally, it should be noted that “systems” of proteins typically include pools
of proteins that define a subproteome, as is common in proteomics and in most
of the chapters of this book. But systems of proteins subjected to proteomic studies
might also include purified/single proteins studied in parallel. That is, for a pool
of N systems-related proteins, one can study: (1) one pool of N-proteins in a
mixture; or (2) N individual proteins in parallel [7]. The latter approach is taken
in the field of structural proteomics, using methods such as nuclear magnetic
resonance (NMR; chapter 17), x-ray crystallography (chapter 18), or even electron
paramagnetic resonance (EPR; chapter 19).
1.4 SUMMARY
The most significant challenge in proteomics is how to detect so many proteins that
cover such wide concentration or dynamic ranges. One solution is to simplify
proteomes into subproteome fractions and then to analyze these. Subproteomes are
defined as proteins related in a systems-based manner so that they can be physically
isolated for study. In this regard, some of the most practical subproteomes for spectral
studies are those associated with organelles (isolated by centrifugation) or those
defined by binding to certain classes of ligands (isolated by affinity chromatography).
FIGURE 1.5 Surfaces used on SELDI chips to select for various subproteomes. Discussed
further in chapter 7.
Biochemical Surfaces for Specific Protein Interaction Studies
Proactivated surface Antibody–antigen Receptor–ligand DNA–protein
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![10 Spectral Techniques in Proteomics
The latter classification is central to the field of chemical proteomics and to most
of the studies presented in this book.
1.5 FUTURE PROSPECTS
The complete systems-based characterization of a proteome will ultimately provide
a description of how that system responds to a biological stimulus such as exposure
to an environmental insult like a pollutant or a drug. Such a complete characterization
of a proteome would also provide an explanation of the underlying biology that
differentiates a disease state from a healthy state. To achieve this goal, the map of
protein interactions in figure 1.1 should be expanded to include protein–ligand or
protein–DNA interactions and should indicate relative levels of the various proteins
as well as the subcellular localization of the proteins involved. This map should also
indicate how the system changes over time after exposure to a biological stimulus.
Ideker et al. [1] have made a significant step towards creating such a comprehensive
proteome map that includes changes in expression levels along with all protein–protein
and protein–DNA interactions associated with galactose use in yeast.
However, more is needed to describe a proteome fully. Such maps could be
extended to include PTMs, levels of mRNA, and levels of metabolites. Improved
spectral tools for analyzing proteomes will be needed to create such maps. Further-
more, the complexity of visualizing and analyzing all of this information has created
a need for improved bioinformatic tools, which are rapidly evolving along with
supporting databases. To help avoid data overload and to coordinate the growing
volume of proteomic data, the Human Proteome Organization (HUPO) has a Web
site to centralize this information: http://www.hupo.org/information/mission.htm.
The goal of monitoring changes in proteomes to identify biological states asso-
ciated with pathology is also important in a practical sense because it permits the
early diagnosis of disease. An exciting advance on the horizon in this regard is the
use of SELDI-MS to profile proteomes (chapter 7) by comparing normal and disease
states and then using these profiles to predict disease. Early successes in predicting
ovarian [21] and prostate [22] cancer have been reported. But before such a technique
can find wide clinical applications [6], certain issues need to be resolved, such as
reproducibility of the profile data (chapter 8) as well as ascertaining the acceptability
of diagnosing based on a profile that involves unknown proteins. It is anticipated
that future advances will address these concerns, along with improvements in the
SELDI separation technology to permit analysis of different subproteome fractions.
The latter would also be important in permitting more comprehensive and faster
chemical proteomic studies. Also, metabonomic data are increasingly used to diag-
nose diseases, with many successes reported in clinical settings (see chapter 16).
Finally, studies of protein–ligand interactions across a proteome are most relevant
if done in the context of a living cell or even a multicellular organism (in vivo). To
this end, recent developments in molecular imaging [23–25] will be an important
complement to in vitro proteomic studies. One exciting advance in this regard is the
use of NMR to provide structural information about protein–ligand interactions
inside living cells [26], as presented in chapter 15.
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![26 Spectral Techniques in Proteomics
3.1 INTRODUCTION
Proteins can be studied with a wide range of spectral techniques, of which only a
small subset are widely use in proteomics. Protein spectral techniques, for the
purposes of this book, are categorized as:
• Mass spectrometry (MS) techniques
• Spectroscopic techniques
Mass spectrometry involves the measurement of protein mass-to-charge (m/z)
ratios, from which molecular weights of intact proteins and their fragments can be
calculated. Spectroscopic techniques all involve monitoring the interaction of electro-
magnetic radiation with matter (table 3.1). Since there are far too many mass
spectrometry and spectroscopic techniques to discuss in a chapter as short as this,
even in a cursory manner, emphasis will be placed on those currently being applied
in proteomics. For more detailed discussion of these methods, the reader is referred
to some of the many excellent books and articles that served as primary sources for
this chapter [1–9].
3.2 MASS SPECTROMETRY [3]
3.2.1 BACKGROUND AND HISTORY
Mass spectrometry was applied to small molecules long before it was used to study
proteins. The first mass spectrometer can be dated to 1912 (J. J. Thompson), with the
first atomic weight measurement made in 1919. The 1950s saw the development of
quadrupole analyzers and the first gas chromatography (GC)-MS. The 1960s saw the
development of tandem MS and electrospray ionization (ESI), with subsequent devel-
opment of liquid chromatography (LC)-MS in 1973 (McLafferty). But MS did not
find broad use for protein studies until the 1980s—first with the development of FAB
(fast atom bombardment) MS in 1981 and then application of ESI to macromolecules
in 1984; these were followed by development of ion cyclotron resonance MS. These
developments opened the door to the application of MS in proteomics in the 1990s,
when protein sequencing with matrix-assisted laser desorption/ionization tandem
mass spectrometry (MALDI MS/MS) was introduced.
Today, MS has evolved to be the most prominent spectral technique used in
proteomic studies. Many technological developments have been and continue to be
made, at an exciting pace. This chapter and this book attempt only to provide a
snapshot in time of some of the more widely used MS techniques and make no
attempt to provide a comprehensive overview of the field. A general description is
provided for the more commonly used mass spectrometers in terms of ionization
method and mass analyzers, since these are the components of greatest variability
and importance in the purchase of an instrument.
3.2.2 IONIZATION METHOD
Proteins must be introduced into the mass spectrometer and ionized so that they can
travel through the mass analyzer and to the detector (fig. 3.1). In the early days of
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![Survey of Spectral Techniques Used to Study Proteins 27
mass spectrometry, EI (electron ionization) was the method of choice for ionization,
but this would fragment the molecule. Such fragmentation provides useful structural
information for small molecules, but makes spectra of proteins overly complex and
uninterpretable. For this reason, softer ionization methods were developed. FAB was
developed first, but had a limited molecular weight range (<7000 g/mol). Later,
MALDI and ESI were developed for protein applications, and these are currently
the most widely used ionization techniques.
3.2.2.1 MALDI
MALDI is described in detail in chapter 5, and elsewhere in this book. In MALDI,
the protein sample is mixed with a solid “matrix” material, then introduced into the
TABLE 3.1
Spectroscopic Techniques Used to Study Proteins
Technique Measured (most common)
Electronic transitions
Fluorescence,a including FPa (fluorescence polarization) and
FRETa (fluorescence resonance energy transfer)
Binding; structure; dynamics
UV-visible (UV-vis) absorbance spectroscopy Electronic structure; binding
CD (circular dichroism) and
MCD (magnetic circular dichroism)
Secondary structure; bonding
Vibrational transitions
IRa (infrared) Structure; bonding
Raman,a resonance Raman,a and polarized Raman Structure; bonding
VCD (vibrational CD) Structure; bonding
Electron and/or nuclear spin transitions
NMRa (nuclear magnetic resonance) Structure and dynamics
CIDNP (chemically induced dynamic nuclear polarization) Structure
EPRb (electron paramagnetic resonance) Structure (paramagnetic)
ENDOR (electron nuclear double resonance) Structure (paramagnetic) and bonding
ESEEM (electron spin-echo envelope modulation) Structure (paramagnetic) and bonding
Other
SPRa (surface plasmon resonance) Binding
X-ray crystallographya Structure
SLS/DLS (static light scattering/dynamic light scattering) Size (mol. wt.; aggregation)
SAXS (small- [or low]-angle x-ray scattering) Size (mol. wt.; aggregation)
Mössbauer and magnetic Mössbauer Bonding (metal)
XAFS/EXAFS (x-ray absorption fine structure/extended x-ray
absorption fine structure)
Bonding (metal)
XANES (x-ray absorption near-edge structure) Bonding (metal)
XAS (x-ray absorption spectroscopy) Bonding (metal)
a Techniques discussed in this chapter and elsewhere in this book.
b Also referred to as ESR (electron spin resonance).
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- 6. SPECTRAL TECHNIQUES IN PROTEOMICS DK3714_C000.fm Page i Friday, February 16, 2007 3:37 PM
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- 8. CRC Press is an imprint of the Taylor & Francis Group, an informa business Boca Raton London New York SPECTRAL TECHNIQUES IN PROTEOMICS Edited by DANIEL S. SEM DK3714_C000.fm Page iii Friday, February 16, 2007 3:37 PM
- 9. CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2007 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 1-57444-580-4 (Hardcover) International Standard Book Number-13: 978-1-57444-580-0 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the conse- quences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www. copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Spectral techniques in proteomics / editor, Daniel S. Sem. p. ; cm. “A CRC title.” Includes bibliographical references and index. ISBN-13: 978-1-57444-580-0 (alk. paper) ISBN-10: 1-57444-580-4 (alk. paper) 1. Proteins--Spectra. 2. Proteomics--Methodology. 3. Mass spectrometry. I. Sem, Daniel S. [DNLM: 1. Proteomics--methods. 2. Mass Spectrometry--methods. 3. Spectrum Analysis--methods. QU 58.5 S741 2007] QP551.S675 2007 572’.633--dc22 2006103310 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com DK3714_C000.fm Page iv Friday, February 16, 2007 3:37 PM
- 10. Dedication In loving thanks to my wife, Teresa, and children, Lucas, Camille, and Isaac, for being a constant source of inspiration and support and for tolerating the countless hours I had to spend immersed in my laptop. DK3714_C000.fm Page v Friday, February 16, 2007 3:37 PM
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- 12. vii Table of Contents Preface.......................................................................................................................xi Editor ......................................................................................................................xiii Contributors ............................................................................................................. xv Abbreviations..........................................................................................................xix PART I The Scope of Proteomic and Chemical Proteomic Studies Chapter 1 The Systems-Based Approach to Proteomics and Chemical Proteomics ........................................................................... 3 Daniel S. Sem Chapter 2 Similarities in Protein Binding Sites................................................. 13 Hugo O. Villar, Mark R. Hansen, and Richard Kho Chapter 3 Survey of Spectral Techniques Used to Study Proteins.................... 25 Daniel S. Sem PART II Mass Spectral Studies of Proteome and Subproteome Mixtures Chapter 4 Capillary Electrophoresis—Mass Spectrometry for Characterization of Peptides and Proteins......................................... 47 Christian Neusüß and Matthias Pelzing Chapter 5 Protein and Peptide Analysis by Matrix-Assisted Laser Desorption/Ionization Tandem Mass Spectrometry (MALDI MS/MS) .............................................................................. 67 Emmanuelle Sachon and Ole Nørregaard Jensen Chapter 6 Characterization of Glycosylated Proteins by Mass Spectrometry Using Microcolumns and Enzymatic Digestion................................ 81 Per Hägglund and Martin R. Larsen DK3714_C000.fm Page vii Friday, February 16, 2007 3:37 PM
- 13. viii Table of Contents Chapter 7 Surface-Enhanced Laser Desorption/Ionization Protein Biochip Technology for Proteomics Research and Assay Development ..... 101 Scot R. Weinberger, Lee Lomas, Eric Fung, and Cynthia Enderwick Chapter 8 An Approach to the Reproducibility of SELDI Profiling............... 133 Walter S. Liggett, Peter E. Barker, Lisa H. Cazares, and O. John Semmes PART III Protein–Protein (or Peptide) Interactions: Studies in Parallel and with Mixtures Chapter 9 Mass Spectrometric Applications in Immunoproteomics ............... 157 Anthony W. Purcell, Nicholas A. Williamson, Andrew I. Webb, and Kim Lau Chapter 10 Near-Infrared Fluorescence Detection of Antigen–Antibody Interactions on Microarrays............................................................. 185 Vehary Sakanyan and Garabet Yeretssian Chapter 11 Application of Shotgun Proteomics to Transcriptional Regulatory Pathways........................................................................ 207 Amber L. Mosley and Michael P. Washburn Chapter 12 Electrophoretic NMR of Protein Mixtures and Its Proteomic Applications............................................................... 223 Qiuhong He, Sunitha B. Thakur, and Jeremy Spater PART IV Chemical Proteomics: Studies of Protein–Ligand Interactions in Pools and Pathways Chapter 13 Characterizing Proteins and Proteomes Using Isotope-Coded Mass Spectrometry........................................................................... 255 Uma Kota and Michael B. Goshe DK3714_C000.fm Page viii Friday, February 16, 2007 3:37 PM
- 14. Table of Contents ix Chapter 14 Surface Plasmon Resonance Biosensors’ Contributions to Proteome Mapping........................................................................... 287 Rebecca L. Rich and David G. Myszka Chapter 15 Application of In-Cell NMR Spectroscopy to Investigation of Protein Behavior and Ligand–Protein Interaction inside Living Cells........................................................................... 305 Volker Dötsch Chapter 16 An Overview of Metabonomics Techniques and Applications....... 321 John C. Lindon, Elaine Holmes, and Jeremy K. Nicholson PART V Structural Proteomics: Parallel Studies of Proteins Chapter 17 NMR-Based Structural Proteomics ................................................. 349 John L. Markley Chapter 18 Leveraging X-Ray Structural Information in Gene Family-Based Drug Discovery: Application to Protein Kinases .... 373 Marc Jacobs, Harmon Zuccola, Brian Hare, Alex Aronov, Al Pierce, and Guy Bemis Chapter 19 EPR Spectroscopy in Genome-Wide Expression Studies............... 391 Richard Cammack PART VI Summary Chapter 20 Summary of Chapters and Future Prospects for Spectral Techniques in Proteomics.................................................. 409 Daniel S. Sem Index...................................................................................................................... 421 DK3714_C000.fm Page ix Friday, February 16, 2007 3:37 PM
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- 16. xi Preface A significant challenge in presenting an overview of Spectral Techniques in Proteomics is in defining the scope of the topic. Proteomics means different things to different people; for years, the dominant technique employed was 2D gel electrophoresis, followed by mass spectrometry (MS). While many exciting MS applications are presented (e.g., matrix-assisted laser desorption/ionization [MALDI], electrospray ionization [ESI], tandem MS, liquid chromatography [LC]-MS, surface-enhanced laser desorption/ionization [SELDI], isotope-coded affinity tag [ICAT]), a comprehen- sive survey of MS methods and applications in proteomics is certainly beyond the scope of this book. Since Spectral Techniques in Proteomics is intended for a broad audience of protein biochemists and biophysicists, topics such as structural proteomics and chemical proteomics will also be covered, along with fluorescence/array-based screening, SPR (surface plasmon resonance), and other “lab-on-a-chip” technologies. Furthermore, a disproportionate amount of time will be spent on some less established spectroscopic methods in proteomics, with forward-looking speculation on future applications. The intention of this book is therefore to facilitate the inno- vation, development, and application of new spectroscopic methods in proteomics while giving a modest overview of existing and proven techniques. To this end, a broader view of proteomics is taken in order to include studies that go beyond the usual scope of 2D gels and MS, attempting to address function and mechanism at the level of protein–ligand interactions. After all, this is the realm in which protein spectroscopists have always excelled and felt most at home. Proteomics is defined broadly as the “systems-based” study of proteins in the organelles, cells, tissues, or organs of an organism. It is the study of the protein complement of the genome in time and space. In practice, this definition sometimes limits proteomics to the study of proteins in 2D gels, since this is one of the few contexts in which so many proteins can be studied at once. It is also possible to simplify a proteome into a more manageable subset (a subproteome) by focusing on a smaller number of proteins related in a systems-based manner. Such systems of interrelated proteins can include: (1) regulatory cascades connected via protein–protein inter- actions; (2) metabolic pathways; (3) proteins with related modifications (acylation, phosphorylation, glycosylation, methylation, etc.); and (4) any collection of proteins associated with a biological effect, such as uncontrolled cell growth (cancer), an immune response, or drug metabolism. This approach to simplifying a proteome into systems-related subproteomes is described in chapter 1. Thus, for purposes of this book, proteomic studies are extended to include the parallel study of subsets of related proteins, some of which were described previously. Such subsets might also include proteins that comprise a unique basis set of protein folds in an organism’s proteome, as currently defines the scope of most structural proteomic initiatives. Another systems-related subset could comprise proteins with DK3714_C000.fm Page xi Friday, February 16, 2007 3:37 PM
- 17. xii Preface similar binding sites (chapter 2), as currently defines the scope of chemical proteomic studies. In this case, the subset of proteins can be considered to be part of a biologically relevant network in the sense that they represent all of the protein–ligand interactions that would occur when an organism is exposed to a given chemical perturbant (drug, pollutant, chemical genetic probe, etc.). One prominent example of such systems-related proteins is those that use the same cofactor or prosthetic group, such as kinases, which all bind ATP (chapter 18). This broader definition of proteomics and systems-based studies is not only a convenience for framing proteome-wide questions, but also has biological relevance. This book aims to provide a broad overview of the spectroscopic toolbox that can be applied in such systems-based studies of proteins, whether they are studied in the context of proteome or subproteome mixtures (traditional proteomics) or as individual/purified proteins studied in parallel (the broader, systems-based view of proteomics), as in structural proteomics. This book begins in part I by defining the scope of the field in order to give coherence to the chapters from the various expert contributors. Proteomics is defined in a way that is relevant to a spectroscopist (chapters 1 and 2) and then a very brief overview of commonly used spectroscopic methods is given (chapter 3). In part II, commonly used MS methods are presented, including separation techniques that typically precede ESI studies, as well as MALDI MS/MS-based protein identifica- tion. SELDI is presented as a tool that combines separation with MS analysis on the same chip. Part III focuses on studies of protein–protein interactions using a variety of techniques, including near-infrared (NIR) fluorescence, nuclear magnetic resonance (NMR), and MS. Part IV covers protein–ligand interactions with tech- niques ranging from MS to SPR to NMR. Recent developments in ICAT labeling strategies are covered, and the section ends with a discussion of metabonomics, since metabolites represent an important functional output of the proteome. Part V covers advances in structural proteomics using NMR, x-ray crystallography, and electron paramagnetic resonance (EPR). The book ends with chapter 20, a summary of current technology and future prospects extracted from the various contributors, again to give added coherence to the topic. Spectral Techniques in Proteomics will be useful for graduate students and other scientists wanting to develop and apply spectroscopic methods in proteomics. It will also be of value to more experienced researchers thinking of moving into this field or those in proteomics looking to broaden the scope of their studies. In short, it is intended for anyone wanting to take a systems-based approach to studying proteins, their function, and their mechanisms using various spectroscopic tools. Daniel Sem DK3714_C000.fm Page xii Friday, February 16, 2007 3:37 PM
- 18. xiii Editor Daniel Sem is an assistant professor in the chemistry department at Marquette University in Milwaukee, Wisconsin. He also serves as director of the Chemical Proteomics Facility at Marquette (CPFM) and is a member of the Marine and Freshwater Biomedical Sciences Center at the University of Wisconsin–Milwaukee in the endocrine disruptor core group. His current research is focused on the devel- opment and application of chemical proteomic probes for the study of protein–ligand interactions. Emphasis is on fluorescence and NMR-based assays, as well as on proteins that are drug targets and proteins that are antitargets, leading to the adverse and toxic side effects of drugs and pollutants. Prior to joining Marquette, Dr. Sem cofounded Triad Therapeutics in San Diego, California, where he served as vice-president for biophysics. In that capacity, he was involved in NMR-based characterization of large protein–ligand complexes, chem- informatic characterization of combinatorial libraries, bioinformatic analysis of gene families, high-throughput screening, and enzymology/assay development. Triad was the first company founded around NMR-driven, structure-based drug design. It had a technology based on a systems-based approach to drug design, targeting gene families of proteins like kinases and dehydrogenases with focused combinatorial chemistry libraries. Dr. Sem graduated from the University of Wisconsin–Milwaukee with a B.S. in chemistry (summa cum laude) and from University of Wisconsin–Madison with a Ph.D. in biochemistry, specializing in mechanistic enzymology. He then pursued postdoctoral studies at McArdle Laboratory for Cancer Research (Madison, Wisconsin), followed by the Scripps Research Institute (La Jolla, California), where he did NMR-based structural biology. He has 20 years of experience using spectral techniques to study protein–ligand interactions in basic and applied research settings. DK3714_C000.fm Page xiii Friday, February 16, 2007 3:37 PM
- 19. DK3714_C000.fm Page xiv Friday, February 16, 2007 3:37 PM
- 20. xv Contributors Alex Aronov Vertex Pharmaceuticals Incorporated Cambridge, Massachusetts Peter E. Barker Biotechnology Division National Institute of Standards and Technology Gaithersburg, Maryland Guy Bemis Vertex Pharmaceuticals Incorporated Cambridge, Massachusetts Richard Cammack Department of Life Sciences Pharmaceutical Sciences Research Division King’s College London, United Kingdom Lisa H. Cazares The Center for Biomedical Proteomics Eastern Virginia Medical School Norfolk, Virginia Volker Dötsch Institute for Biophysical Chemistry Center for Biomolecular Magnetic Resonance University of Frankfurt Frankfurt, Germany Cynthia Enderwick Ciphergen Biosystems Inc. Fremont, California Eric Fung Ciphergen Biosystems Inc. Fremont, California Michael B. Goshe Department of Molecular and Structural Biochemistry North Carolina State University Raleigh, North Carolina Per Hägglund Biochemistry and Nutrition Group Technical University of Denmark Lyngby, Denmark Mark R. Hansen Altoris, Inc. San Diego, California Brian Hare Vertex Pharmaceuticals Incorporated Cambridge, Massachusetts Qiuhong He Departments of Radiology and Bioengineering University of Pittsburgh Cancer Institute MR Research Center, University of Pittsburgh Pittsburgh, Pennsylvania Elaine Holmes Department of Biomolecular Medicine Faculty of Medicine Imperial College London South Kensington, London, United Kingdom DK3714_C000.fm Page xv Friday, February 16, 2007 3:37 PM
- 21. xvi Contributors Marc Jacobs Vertex Pharmaceuticals Incorporated Cambridge, Massachusetts Ole Nørregaard Jensen Protein Research Group Department of Biochemistry and Molecular Biology University of Southern Denmark Odense, Denmark Richard Kho Altoris, Inc. San Diego, California Uma Kota Department of Molecular and Structural Biochemistry North Carolina State University Raleigh, North Carolina Martin R. Larsen Department of Biochemistry and Molecular Biology University of Southern Denmark Odense, Denmark Kim Lau Department of Biochemistry and Molecular Biology Bio21 Molecular Science and Biotechnology Institute University of Melbourne Victoria, Australia Walter S. Liggett Statistical Engineering Division National Institute of Standards and Technology Gaithersburg, Maryland John C. Lindon Department of Biomolecular Medicine Faculty of Medicine Imperial College London South Kensington, London, United Kingdom Lee Lomas Ciphergen Biosystems Inc. Fremont, California John L. Markley Center for Eukaryotic Structural Genomics National Magnetic Resonance Facility at Madison Biochemistry Department University of Wisconsin–Madison Madison, Wisconsin Amber L. Mosley Stowers Institute for Medical Research Kansas City, Missouri David G. Myszka Center for Biomolecular Interaction Analysis School of Medicine University of Utah Salt Lake City, Utah Christian Neusüß Bruker Daltonik GmbH Leipzig, Germany Jeremy K. Nicholson Department of Biomolecular Medicine Faculty of Medicine Imperial College London South Kensington, London, United Kingdom DK3714_C000.fm Page xvi Friday, February 16, 2007 3:37 PM
- 22. Contributors xvii Matthias Pelzing Bruker Daltonik GmbH Leipzig, Germany Al Pierce Vertex Pharmaceuticals Incorporated Cambridge, Massachusetts Anthony W. Purcell Department of Biochemistry and Molecular Biology Bio21 Molecular Science and Biotechnology Institute University of Melbourne Victoria, Australia Rebecca L. Rich Center for Biomolecular Interaction Analysis School of Medicine University of Utah Salt Lake City, Utah Emmanuelle Sachon Protein Research Group Department of Biochemistry and Molecular Biology University of Southern Denmark Odense, Denmark Daniel S. Sem Chemical Proteomics Facility at Marquette Department of Chemistry Marquette University Milwaukee, Wisconsin O. John Semmes The Center for Biomedical Proteomics Eastern Virginia Medical School Norfolk, Virginia Jeremy Spater Departments of Radiology and Bioengineering University of Pittsburgh Cancer Institute MR Research Center University of Pittsburgh Pittsburgh, Pennsylvania Sunitha B. Thakur Departments of Radiology and Bioengineering University of Pittsburgh Cancer Institute MR Research Center University of Pittsburgh Pittsburgh, Pennsylvania Sakanyan Vehary ProtNeteomix Université de Nantes Nantes, France Hugo O. Villar Altoris, Inc. San Diego, California Michael P. Washburn Stowers Institute for Medical Research Kansas City, Missouri Andrew I. Webb Department of Biochemistry and Molecular Biology Bio21 Molecular Science and Biotechnology Institute University of Melbourne Victoria, Australia Scot R. Weinberger GenNext Technologies™ Montara, California DK3714_C000.fm Page xvii Friday, February 16, 2007 3:37 PM
- 23. xviii Contributors Nicholas A. Williamson Department of Biochemistry and Molecular Biology Bio21 Molecular Science and Biotechnology Institute University of Melbourne Victoria, Australia Garabet Yeretssian Biotechnologie, Biocatalyse, Biorégulation Faculté des Sciences et des Techniques Université de Nantes Nantes, France Harmon Zuccola Vertex Pharmaceuticals Incorporated Cambridge, Massachusetts DK3714_C000.fm Page xviii Friday, February 16, 2007 3:37 PM
- 24. xix Abbreviations 2D two dimensional AAs amino acids ALICE acid-labile isotope-coded extractants ANTS 8-aminonaphthalene-1,3,6-trisulfonate APC antigen presenting cell APCI atmospheric pressure chemical ionization APTA (3-acrylamidopropyl)-trimethylammonium chloride APPI atmospheric pressure photo ionization AQUA absolute quantification ATP adenosine triphosphate BLAST basic local alignment search tool BSA bovine serum albumin CA-ENMR capillary-array ENMR CBB Coomassie Brilliant Blue CCA canonical correlation analysis CC-ENMR convection-compensated ENMR CD circular dichroism CDK cyclin-dependent kinase CE capillary electrophoresis CEC capillary electrochromatography CESG Center for Eukaryotic Structural Genomics CGE capillary gel electrophoresis CHD coronary heart disease CID collision-induced dissociation CIEF capillary isoelectric focusing CLOUDS classification of unknowns by density superposition COMET Consortium for Metabonomic Toxicology CORES complexes restricted by experimental structures COSY correlation spectroscopy CRINEPT cross relaxation-enhanced polarization transfer CSF cerebrospinal fluid CT constant time CTL cytotoxic T lymphocyte CZE capillary zone electrophoresis DA discriminant analysis DCs dendritic cells DC direct current DHB 2,5-dihydroxybenzoic acid DK3714_C000.fm Page xix Friday, February 16, 2007 3:37 PM
- 25. xx Abbreviations DisProt Database of Protein Disorder ECD electron capture dissociation EGFR epidermal growth factor receptor EI electron ionization EIE extracted ion electropherogram ELISA enzyme-linked immunosorption assay ENMR electrophoretic NMR EOF electro-osmotic flow EPR electron paramagnetic resonance ESI electrospray ionization ESR electron spin resonance FAB fast atom bombardment FP fluorescence polarization FRET fluorescence resonance energy transfer FT Fourier transform FTICR Fourier transform ion cyclotron resonance GC-MS gas chromatography-mass spectrometry GFP green fluorescent protein GIST global internal standard technology GlcNAc N-acetylglucosamine GPCR G-protein coupled receptor GPI glycosyl-phosphatidylinositol GST glutathione-S-transferase HCCA alpha-cyano-4-hydroxycinnamic acid HILIC hydrophilic interaction liquid chromatography HPLC high-performance liquid chromatography HSQC heteronuclear single quantum coherence HTS high-throughput screening ICAT isotope-coded affinity tag ICGs interchromatin granules IEF isoelectric focusing Ig immunoglobulin IGOT isotope-coded glycosylation site-specific tagging IMAC immobilized metal affinity chromatography INEPT insensitive nuclei enhanced by polarisation transfer IR infrared Irk insulin receptor kinase IRMPD infrared multiphoton photodissociation IT ion trap ITP isotachophoresis LC liquid chromatography LC/MS/MS liquid chromatography tandem mass spectrometry LCM laser capture microdissection LDI laser desorption/ionization LDL low-density lipoprotein LIF laser-induced fluorescence DK3714_C000.fm Page xx Friday, February 16, 2007 3:37 PM
- 26. Abbreviations xxi LIMS laboratory information management systems LLE liquid–liquid extraction mAb monoclonal antibody MALDI matrix-assisted laser desorption/ionization MAS magic angle spinning MCAT mass-coded abundance tagging MEKC micellar electrokinetic chromatography MEM maximum entropy method MHC major histocompatibility complex MS mass spectrometry MS/MS tandem mass spectrometry MudPIT multidimensional protein identification technology MW molecular weight NACE-MS nonaqueous CE-MS NIR near infrared NMR nuclear magnetic resonance NMRFAM National Magnetic Resonance Facility at Madison NOE nuclear Overhauser effect NPC nuclear pore complex PAGE polyacrylamide gel electrophoresis PC phosphatidylcholine PC principal component PCA principal components analysis PCR polymerase chain reaction PDB Protein Data Bank PECAN protein energetic conformational analysis from NMR chemical shifts PhIAT phosphoprotein isotope-coded affinity tag PhIST phosphoprotein isotope-coded solid-phase tag PI-PLC phosphatidylinositol phospholipase C PISTACHIO probabilistic identification of spin systems and their assignments including coil-helix inference as output PKA c-AMP dependent kinase (protein kinase A) PLS partial least squares PML promyelocytic leukemia PTM post-translational modification Q quadrupolar (as in Q-TOF) QC quality control QD quantum dot QqQ triple quadrupole QUEST quantitation using enhanced signal tags RCSB Research Collaboratory for Structural Biology RDCs residual dipolar couplings RMSD root mean square deviation RP reverse phase RPLC reverse phase liquid chromatography RR resonance Raman DK3714_C000.fm Page xxi Friday, February 16, 2007 3:37 PM
- 27. xxii Abbreviations SA sinapinic acid SAX strong anion exchange SBDD structure-based drug design SCX strong cation exchange SDS sodium dodecyl sulfate SEAC surface-enhanced affinity capture SELDI surface-enhanced laser desorption/ionization SEND surface-enhanced neat desorption SEREX serological expression of cDNA expression libraries SILAC stable isotope labeling by amino acids in cell culture SMRS standard metabolic reporting structures SPE solid phase extraction SPITC 4-sulfophenyl isothiocynate SPR surface plasmon resonance STE stimulated echo TAP tandem affinity purification TcR T cell receptor TLF time-lag focusing TFA trifluoroacetic acid TOF time of flight TROSY transverse relaxation optimized spectroscopy TSP 3-(trimethylsilyl) propionic 2,2,3,3-d4 acid VICAT visible isotope-coded affinity tag VLDL very low density lipoprotein DK3714_C000.fm Page xxii Friday, February 16, 2007 3:37 PM
- 28. Part I The Scope of Proteomic and Chemical Proteomic Studies DK3714_S001.fm Page 1 Wednesday, November 8, 2006 4:13 PM
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- 30. 3 1 The Systems-Based Approach to Proteomics and Chemical Proteomics Daniel S. Sem CONTENTS 1.1 Introduction...................................................................................................... 3 1.2 Complexity and Dynamic Range Challenges.................................................. 4 1.3 The Systems-Based Approach ......................................................................... 4 1.3.1 Systems-Based Relationships .............................................................. 4 1.3.2 Subproteomes....................................................................................... 6 1.3.3 Chemical Proteomics ........................................................................... 7 1.3.4 Applications ......................................................................................... 8 1.4 Summary .......................................................................................................... 9 1.5 Future Prospects............................................................................................. 10 References................................................................................................................ 11 1.1 INTRODUCTION Simply stated, proteomics is the study of the protein complement of a genome using the tools of protein biochemistry on a proteome-wide scale. It is devoted to monitoring changes in expression levels or post-translational modifications of all the proteins in an organism, organ, cell, or organelle as a function of time or biological state (e.g., diseased vs. healthy). Ideally, it should also address protein structure–function in terms of interactions with substrates, drugs, inhibitors, lipids, DNA, or other proteins. It is possible to infer some information about protein expression levels based on changes in mRNA detected using microarray technology—an elegant coupling of microfluidics, “lab-on-a-chip,” and detection (usually fluorescence-based) technolo- gies. But, mRNA levels are not always correlated well with protein levels, and they reveal nothing about post-translational modification or protein interactions. As such, the field of proteomics serves an essential function, despite the additional technical challenges involved in analyzing proteins in comparison with polynucleotides [1–4]. Most significant is the challenge of dealing with sample complexity and dynamic range. DK3714_C001.fm Page 3 Monday, February 5, 2007 12:03 PM
- 31. 4 Spectral Techniques in Proteomics 1.2 COMPLEXITY AND DYNAMIC RANGE CHALLENGES The human genome comprises over 30,000 genes, which encode many more proteins; many are variants due to alternative splicing and PTMs (post-translational modifica- tions). Over 400 PTMs are known to date, so there is tremendous complexity in the proteome as it is expressed in a given cell type. While it is a challenge to resolve the thousands of proteins expressed in a proteome, it is an even greater challenge because these proteins may be present at very different concentrations, ranging over six to nine orders of magnitude depending on the cell type. For example, serum contains albumin as the most abundant protein (at ~40 mg/mL and ~50% of blood protein), while other proteins of interest, such as interleukin-6, are present at <5 pg/mL [5]. The need to quantify so many proteins over such a wide concentration range is one of the greatest challenges in proteomics. Other challenges include the need to assess interactions between the proteins and their various ligands that define biological networks or systems. Furthermore, at least in some cases, these interactions should also be measured within a cell (chapter 15) to ensure their biological relevance in the context of potential accessory proteins or cofactors, as well as under physiologically relevant conditions (water activity, pH, ionic strength, lipids, etc.). 1.3 THE SYSTEMS-BASED APPROACH 1.3.1 SYSTEMS-BASED RELATIONSHIPS Proteomics is considered a subdiscipline of systems biology. So what is systems biology? Weston and Hood define it as “the analysis of the relationships between elements in a system in response to genetic or environmental perturbations, with the goal of understanding the system or the emergent properties of the system” [6]. “System” is a broad term borrowed from other fields (e.g., engineering), but in a biological context the word usually refers to organelles, cells, organs, or organisms. Such a definition is therefore based largely on the physical location of the proteins studied, their network of interactions, and their collective role in defining a bio- logical entity (such as a mitochondrion or a liver cell) or function (such as the immune response). It is also possible to define systems at the level of molecules based on the network of interactions that occur between them, usually within the context of a single cell or organelle. This typically involves the measurement of all pairs of protein–protein interactions that can occur, using techniques such as the yeast two-hybrid system. In this manner it is possible to establish networks of proteins that participate in interactions with each other. These pairs of interactions, summed across a whole proteome, comprise a protein interaction map such as that shown in figure 1.1 and discussed in chapter 14 by Rich and Myszka. In a broad sense, systems or networks of interacting proteins can be defined based on the presence of interactions between [7]: (a) Two proteins directly, as in a regulatory cascade when a protein kinase phosphorylates another protein DK3714_C001.fm Page 4 Monday, February 5, 2007 12:03 PM
- 32. The Systems-Based Approach to Proteomics and Chemical Proteomics 5 (b) Two proteins that are sequential enzymes in a metabolic pathway, whereby they are related by binding to a common ligand that is a substrate for one enzyme and a product for another (c) Two proteins that bind to a common ligand, such as a nonspecific drug that binds to multiple targets (as is common for protein kinase inhibitors) or even a cofactor (NAD(P)H binds to all dehydrogenases) FIGURE 1.1 Systems relationships in a so-called “protein interaction map” for Drosophilia melanogaster. Proteins are coded by subcellular location as well as interactions (indicated with lines). Most probable interactions are indicated with darker lines. (Reprinted from Giot, L. et al., Science, 302, 1727–1736, 2003. With permission from the American Associa- tion for the Advancement of Science, 2003, and discussed further in chapter 14.) Sub-Cellular Localization View Extracellular Extracellular matrix Plasma membrane Synaptic vesicle Mitochondria Endoplasmic reticulum Golgi Lysosome Cytoplasm Cytoskeleton Peroxisome Ribosome Centrosome Nucleus Unknown Nuclear proteins Cytoplasmic proteins Interaction Ratings 0.9–1.0 0.8–0.9 0.65–0.8 <0.65 Membrane and extracellular proteins DK3714_C001.fm Page 5 Monday, February 5, 2007 12:03 PM
- 33. 6 Spectral Techniques in Proteomics These interactions are defined schematically in figure 1.2 and are central to defining the field of chemical proteomics. 1.3.2 SUBPROTEOMES To simplify the complexity of a proteome in order to make proteomic studies more manageable, it is common to create subproteomes—that is, to study a subset of the whole proteome, where that subset is defined based on some systems-based rela- tionship between proteins. Proteins can be grouped into subproteomes in the many ways mentioned in the previous sections. These include: 1. location within the cell (e.g., golgi, lysosome, nucleus) 2. participation in a large functionally defined protein complex (e.g., ribosome, transcription initiation complex, cytoskeleton) 3. shared post-translational processing (e.g., phosphorylation, glycosylation) 4. affinity for a ligand (e.g., ATP, NAD(P)H, drugs) 5. chemically reactive groups (e.g., cysteine thiols, lysine amines) 6. shared biological function (e.g., immunoproteome) These groupings are based on physical location in the cell, chemical properties (ligand binding, PTM), or functional role. Classifications were made based on practical considerations in that each provides a means to isolate the subproteome, although it is often not possible to isolate category 6 subproteomes. Systems defined by networks of protein–protein interactions, as identified in figure 1.1, are often not easy to isolate. FIGURE 1.2 Systems relationships that are relevant in proteomics and chemical proteomics. Proteins can be related by: (a) metabolic pathway, with protein pairs related by binding to the same molecule, which is a substrate for one enzyme and a product for another; (b) ligand binding, where protein pairs are related by binding the same ligand (not necessarily enzyme and substrate); or (c) regulatory cascade, where proteins interact directly with each other, as when a protein kinase phosphorylates another protein. (Adapted from Sem, D.S., Expert Rev. Proteomics, 1, 165–178, 2004.) E2 A (b) E3 E1 E1A E3A E2A A (a) B C D E1 E2 E3 E2 E3 (c) E∗ 1 E1 DK3714_C001.fm Page 6 Monday, February 5, 2007 12:03 PM
- 34. The Systems-Based Approach to Proteomics and Chemical Proteomics 7 Subproteomes categorized based on physical location in the cell are the easiest to define since separation techniques permit isolation of organelles [8]. The most general breakdown of cellular location is into the nucleus, cytoplasm, or membrane/ extracellular region. A more detailed breakdown would include the 14 categories of subcellular locations and organelles defined in figure 1.1 [9]. Subproteomes defined based on protein–ligand interactions are presented at greater length later in this book (part IV) and fall largely within the scope of chemical proteomics, described in the next section. 1.3.3 CHEMICAL PROTEOMICS Chemical proteomics is a branch of proteomics [7,10–14] with a focus on directly detected protein–ligand interactions measured across a systems-related group of proteins. It is therefore a mechanistic complement to chemical genetics. In chemical genetics, the observable is a phenotypic change induced by a chemical knockout [15], but without any direct characterization of protein–ligand interactions. Bogyo defined chemical proteomics as being focused on the structure, function, and role of proteins in different biological systems using chemical probes [10]. Thus, the field relies heavily on chemical probes to define systems-related proteins. These probes can be activity based (covalently labeling all proteins that share a common electrophile or nucleophile), as illustrated in the left side of figure 1.3. An example might be labeling of all thiol groups, as done with the ICAT (isotope- coded affinity tag) technology pioneered byAebersold [16] and presented in chapter 13. FIGURE 1.3 Chemical proteomic studies. Such studies employ probes to “profile” proteins. This can be done using: (a) activity-based profiling, with probes that covalently label proteins (left arrow); or (b) affinity-based profiling, with probes that noncovalently bind to proteins (right panel). (Adapted from Sem, D.S., Expert Rev. Proteomics, 1, 165–178, 2004.) –OH –S– Proteome or Sub-proteome Proteins related by: (a) Binding site shape or (b) Binding site nucleophile or electrophile –OH –OH –OH –S– –OH –OH –S –S –S– –S– DK3714_C001.fm Page 7 Monday, February 5, 2007 12:03 PM
- 35. 8 Spectral Techniques in Proteomics Detection of probes can also take place by using fluorescence, as developed by Bogyo and others [10,17]. Probes can also be affinity based (right side of figure 1.3), where they bind noncovalently to families of proteins [7,12,18] with related binding sites (so-called pharmaco families [14,19,20]). Many subproteomes are isolated and characterized based on common binding sites, so such classification is extremely important for the fields of proteomics and chemical proteomics. It is also important in drug design, where binding site similarities can determine how specific a drug is for its intended protein target relative to antitargets, which include drug-metabolizing enzymes as well as proteins that produce undesired side effects. The science of classifying protein binding sites based on ligand-binding preferences is a growing field that has evolved independently of proteomics. It is discussed in detail in chapter 2 by Villar et al. 1.3.4 APPLICATIONS Most proteomic studies now employ mass spectral detection, usually of proteins extracted from 2D gels (chapter 4) or fluorescence studies of microarrays (chapter 10 and fig. 1.4). An alternative to matrix-assisted laser desorption/ionization (MALDI) analysis of extracted proteins is to use in-line purification of proteins by capillary electrophoresis (CE), followed by electrospray ionization-mass spectrometry (ESI-MS) (chapter 4). Although these methods provide good resolution, to help address the complexity problem in proteomics, they usually also require further simplification of proteome samples. The systems-based approaches described earlier for simplify- ing proteomes are therefore crucial in most proteome studies. In particular, it is FIGURE 1.4 Various subproteomes quantified using fluorescence detection of microarrays. Different ways of capturing subproteomes are shown using antibodies, antigens, ligands, and other affinity–capture arrays. Discussed further in chapter 10. Antibody array Antigen array Micro & microarrays Fluorescence detection Binding Labeling Antibody Ligand Peptide Protein Phage displayed array Total protein array DK3714_C001.fm Page 8 Monday, February 5, 2007 12:03 PM
- 36. The Systems-Based Approach to Proteomics and Chemical Proteomics 9 routine to analyze subproteomes rather than entire proteomes. This book presents studies of many different subproteomes, including: • immunoproteomes (chapters 9, 10) • glycoproteomes (chapters 6, 13) • phosphoproteomes (chapter 13) • transcriptional regulatory pathways (chapter 11) • ATP-binding proteins/protein kinases (chapter 18) • the metabonomes (chapter 16) Many of these studies require some technique to first purify the desired subpro- teome, usually based on an affinity purification step before MS analysis. As an alternative, surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-MS; chapter 7) employs affinity purification on the chip itself, which is coated with an appropriate ligand that captures the desired subproteome (fig. 1.5). Related affinity-capture techniques are also possible using microarrays (fig. 1.4). Finally, it should be noted that “systems” of proteins typically include pools of proteins that define a subproteome, as is common in proteomics and in most of the chapters of this book. But systems of proteins subjected to proteomic studies might also include purified/single proteins studied in parallel. That is, for a pool of N systems-related proteins, one can study: (1) one pool of N-proteins in a mixture; or (2) N individual proteins in parallel [7]. The latter approach is taken in the field of structural proteomics, using methods such as nuclear magnetic resonance (NMR; chapter 17), x-ray crystallography (chapter 18), or even electron paramagnetic resonance (EPR; chapter 19). 1.4 SUMMARY The most significant challenge in proteomics is how to detect so many proteins that cover such wide concentration or dynamic ranges. One solution is to simplify proteomes into subproteome fractions and then to analyze these. Subproteomes are defined as proteins related in a systems-based manner so that they can be physically isolated for study. In this regard, some of the most practical subproteomes for spectral studies are those associated with organelles (isolated by centrifugation) or those defined by binding to certain classes of ligands (isolated by affinity chromatography). FIGURE 1.5 Surfaces used on SELDI chips to select for various subproteomes. Discussed further in chapter 7. Biochemical Surfaces for Specific Protein Interaction Studies Proactivated surface Antibody–antigen Receptor–ligand DNA–protein DK3714_C001.fm Page 9 Monday, February 5, 2007 12:03 PM
- 37. 10 Spectral Techniques in Proteomics The latter classification is central to the field of chemical proteomics and to most of the studies presented in this book. 1.5 FUTURE PROSPECTS The complete systems-based characterization of a proteome will ultimately provide a description of how that system responds to a biological stimulus such as exposure to an environmental insult like a pollutant or a drug. Such a complete characterization of a proteome would also provide an explanation of the underlying biology that differentiates a disease state from a healthy state. To achieve this goal, the map of protein interactions in figure 1.1 should be expanded to include protein–ligand or protein–DNA interactions and should indicate relative levels of the various proteins as well as the subcellular localization of the proteins involved. This map should also indicate how the system changes over time after exposure to a biological stimulus. Ideker et al. [1] have made a significant step towards creating such a comprehensive proteome map that includes changes in expression levels along with all protein–protein and protein–DNA interactions associated with galactose use in yeast. However, more is needed to describe a proteome fully. Such maps could be extended to include PTMs, levels of mRNA, and levels of metabolites. Improved spectral tools for analyzing proteomes will be needed to create such maps. Further- more, the complexity of visualizing and analyzing all of this information has created a need for improved bioinformatic tools, which are rapidly evolving along with supporting databases. To help avoid data overload and to coordinate the growing volume of proteomic data, the Human Proteome Organization (HUPO) has a Web site to centralize this information: http://www.hupo.org/information/mission.htm. The goal of monitoring changes in proteomes to identify biological states asso- ciated with pathology is also important in a practical sense because it permits the early diagnosis of disease. An exciting advance on the horizon in this regard is the use of SELDI-MS to profile proteomes (chapter 7) by comparing normal and disease states and then using these profiles to predict disease. Early successes in predicting ovarian [21] and prostate [22] cancer have been reported. But before such a technique can find wide clinical applications [6], certain issues need to be resolved, such as reproducibility of the profile data (chapter 8) as well as ascertaining the acceptability of diagnosing based on a profile that involves unknown proteins. It is anticipated that future advances will address these concerns, along with improvements in the SELDI separation technology to permit analysis of different subproteome fractions. The latter would also be important in permitting more comprehensive and faster chemical proteomic studies. Also, metabonomic data are increasingly used to diag- nose diseases, with many successes reported in clinical settings (see chapter 16). Finally, studies of protein–ligand interactions across a proteome are most relevant if done in the context of a living cell or even a multicellular organism (in vivo). To this end, recent developments in molecular imaging [23–25] will be an important complement to in vitro proteomic studies. One exciting advance in this regard is the use of NMR to provide structural information about protein–ligand interactions inside living cells [26], as presented in chapter 15. DK3714_C001.fm Page 10 Monday, February 5, 2007 12:03 PM
- 38. The Systems-Based Approach to Proteomics and Chemical Proteomics 11 REFERENCES 1. Ideker, T., Thorsson, V., Ranish, J.A., Christmas, R., Buhler, J., Eng, J.K., Bumgarner, R., Goodlett, D.R., Aebersold, R., and Hood, L., Integrated genomic and proteomic analyses of a systematically perturbed metabolic network, Science, 292, 929–934, 2001. 2. Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H., and Aebersold, R., Quanti- tative analysis of complex protein mixtures using isotope-coded affinity tags, Nat. Biotechnol., 17, 994–999, 1999. 3. Griffin, T.J., Gygi, S.P., Ideker, T., Rist, B., Eng, J., Hood, L., and Aebersold, R., Complementary profiling of gene expression at the transcriptome and proteome levels in Saccharomyces cerevisiae, Mol. Cell. Proteomics, 1, 323–333, 2002. 4. Baliga, N.S., Pan, M., Goo, Y.A., Yi,E.C., Goodlett, D.R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W.V., and Hood, L., Coordinate regulation of energy transduction modules in Halobacterium sp. analyzed by a global systems approach, Proc. Natl. Acad. Sci. U.S.A., 99, 14913–14918, 2002. 5. Anderson, N.L., and Anderson, N.G., The human plasma proteome: history, character, and diagnostic prospects, Mol. Cell. Proteomics, 1, 845–867, 2002. 6. Weston, A.D., and Hood, L.H., Systems biology, proteomics, and the future of health care: toward predictive, preventative, and personalized medicine, J. Proteome Res., 3, 179–196, 2004. 7. Sem, D.S., Chemical proteomics from an NMR spectroscopy perspective, Expert Rev. Proteomics, 1, 165–178, 2004. 8. Wilson, K., Walker, J., and Wilson, J.M., Principles and Techniques of Practical Biochemistry, Cambridge University Press, New York, 2000. 9. Giot, L. et al., A protein interaction map of Drosophila melanogaster, Science, 302, 1727–1736, 2003. 10. Jeffery D., and Bogyo, M., Chemical proteomics and its application to drug discovery, Curr. Opin. Biotechnol., 11, 602–609, 2000. 11. Adam, G.C., Sorensen, E.J., and Cravatt, B.F., Chemical strategies for functional proteomics, Mol. Cell. Proteomics, 1, 781–790, 2002. 12. Pullela, P.K., and Sem, D.S., NMR-driven chemical proteomics: The functional and mechanistic complement to proteomics. In Separation Methods in Proteomics, ed. Smejkal, G.B. and Lazarev, A., CRC Press, Boca Raton, FL, 467–487, 2006. 13. Sem, D.S., Villar, H., and Kelly, M., NMR on target, Mod. Drug Discovery, August 26–31, 2003. 14. Sem, D.S. et al., Systems-based design of bi-ligand inhibitors of oxidoreductases: Filling the chemical proteomic toolbox, Chem. Biol., 11, 185–194, 2004. 15. Peterson, R.T., Link, B.A., Dowling, J.E., and Schreiber, S.L., Small molecule devel- opmental screens reveal the logic and timing of vertebrate development, Proc. Natl. Acad. Sci. USA, 97, 12965–12969, 2000. 16. Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H., and Aebersold, R. Quanti- tative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol., 17, 994–999, 1999. 17. Greenbaum, D., Medzihradszky, K.F., Burlingame, A., and Bogyo, M., Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem. Biol., 7, 569–581, 2000. 18. Yao, H., and Sem, D.S., Cofactor fingerprinting with STD NMR to characterize proteins of unknown function: Identification of a rare cCMP cofactor preference, FEBS Lett., 579, 661–666, 2005. DK3714_C001.fm Page 11 Monday, February 5, 2007 12:03 PM
- 39. 12 Spectral Techniques in Proteomics 19. Kho, R. et al., A path from primary protein sequence to ligand recognition, Proteins, 50, 589–599, 2003. 20. Kho, R. et al., Genome-wide profile of oxidoreductases in viruses, prokaryotes, and eukaryotes, J. Proteome Res., 2, 626–632, 2003. 21. Petricoin, E.F. et al., Use of proteomic patterns in serum to identify ovarian cancer, Lancet, 359, 572–577, 2002. 22. Petricoin, E.F., III, et al., Serum proteomic patterns for detection of prostate cancer, J. Natl. Cancer Inst., 94, 1576–1578, 2002. 23. Meade, T.J., Taylor, A.K., and Bull, S.R., New magnetic resonance contrast agents as biochemical reporters, Curr. Opin. Neurobiol., 13, 597–602, 2003. 24. Nivorozhkin, A.L. et al., Enzyme-activated Gd3+ magnetic resonance imaging contrast agents with a prominent receptor-induced magnetization enhancement, Angew. Chem. Int. Ed. Engl., 40, 2903–2906, 2001. 25. Costache, A.D., Pullela, P.K., Kasha, P., Tomasiewicz, H., and Sem, D.S., Homology- modeled ligand-binding domains of zebrafish estrogen receptors α, β1 and β2: From in silico to in vivo studies of estrogen interactions in Danio rerio as a model system, J. Molecular Endocrinol., 19, 2979–2990, 2005. 26. Serber, Z., Ledwidge, R., Miller, S.M., and Dotsch, V., Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectro- scopy, J. Am. Chem. Soc., 123, 8895–8901, 2001. DK3714_C001.fm Page 12 Monday, February 5, 2007 12:03 PM
- 40. 13 2 Similarities in Protein Binding Sites Hugo O. Villar, Mark R. Hansen, and Richard Kho CONTENTS 2.1 Introduction.................................................................................................... 13 2.2 The Process of Ligand Recognition .............................................................. 14 2.3 Amino-Acid Preference in Binding Sites...................................................... 15 2.4 Conserved Sequence and Structural Motifs at Binding Sites....................... 16 2.5 Three-Dimensional Descriptors for Binding Site Characterization.............. 18 2.6 Experimental Evidence of Binding Similarities............................................ 19 2.7 Exploiting the similarities in Protein Binding Sites: Modular Approaches to Drug Design............................................................ 20 2.8 Future Prospects............................................................................................. 21 References................................................................................................................ 22 2.1 INTRODUCTION Each protein is unique. Each has a unique primary sequence, folds into a unique tertiary structure, and carries out a unique set of functions. Despite each protein’s uniqueness, there are commonalities that can be observed across even the most diverse set of proteins. The conserved aspects of protein structure and function have generated fundamental insights, some of which may have important implications for drug discovery. We are particularly interested in the features that are conserved in protein binding sites because they can have implications for the development of novel techniques in drug design.1 The success of fragment-based2 inhibitor design, popular in methods that use nuclear magnetic resonance (NMR), depends on the cross-reactivity of scaffolds that can span the diversity of protein binding sites.3 If no similarities were found across proteins, then it stands to reason that the libraries would be much larger than if similarities were found. Conserved features are also important for proteomics because they allow the classification of proteins and consequently provide a framework for the organization of the proteome. In our case, the classification we seek is at the interface with chemistry, where patterns of ligand binding can be used to group or differentiate proteins, giving rise to the subfield of chemoproteomics. DK3714_C002.fm Page 13 Monday, February 5, 2007 12:04 PM
- 41. 14 Spectral Techniques in Proteomics The fact that conserved features exist in proteins can be deduced from a large number of indirect observations. For example, all small molecular weight drugs have side effects, most of which are mediated via interactions with proteins other than the intended target. Also, those molecules are metabolized by proteins other than the target and therefore proteins involved in transport or metabolism should also share some similarities with the protein target. For quite some time,4 the literature has reported that small molecules can commonly bind to multiple proteins. The converse is true as well: A single protein can interact with a variety of chemicals, even though they may have little resemblance to each other. The process of small-molecule recognition by a protein is complex and should not be oversimplified.5 Most of our observations and insights gained through the years have been due to the data coming from x-ray crystallography. However, these data provide only a snapshot of the binding process and do not present the dynamic aspects, which may play a significant role. Other spectroscopic techniques are essential to provide complementary information to develop a more thorough under- standing of the interaction process. This chapter will provide a broad but not comprehensive overview of some of the current state of the art in protein binding site characterization, ligand recognition, and classification of proteins based on binding sites. The results are presented in the context of small-molecule drug design, with two major emphases. The first is with respect to the role of similarities in protein binding sites in modular approaches for drug discovery. The second is on how these similarities are incorporated into tech- nologies and information mining strategies to take advantage of the ever increasing amount of data on protein–ligand interactions and binding site characterization. 2.2 THE PROCESS OF LIGAND RECOGNITION The first theoretical models of small-molecule protein interaction were based on the lock and key concept. Despite its simplicity, Fischer’s theory accurately describes the need to have complementarities between the protein and the ligand.6 Unlike locks and keys, however, ligand and protein are dynamic entities. The number of cross- interactions between proteins and ligands are problematic for the lock and key model because small molecules and proteins are promiscuous; a key can fit multiple locks and a lock can accept a variety of keys. The induced-fit model proposed by Koshland appears more reasonable in this respect6 because it views the process as an adaptation of the structures of the ligand and the receptor to each other. Since the proteins are adapting to the ligand and vice versa, it is possible to see how promiscuous compounds and proteins may arise. The lock and key model can then be viewed as a snapshot of the induced fit that occurs when the protein and ligand conformations are the same in the unbound states as in the bound state. Proteins are not in a single static conformation, but rather in a statistical ensemble in thermodynamic equilibrium. NMR and other spectroscopic techniques7–10 corroborate this idea, first introduced by Straub.11 Because the proteins are in equilibrium among a number of preexisting conformations, displacement of the equilibrium can occur upon ligand binding, towards the protein conformer that has the most favorable interactions with the ligands. DK3714_C002.fm Page 14 Monday, February 5, 2007 12:04 PM
- 42. Similarities in Protein Binding Sites 15 The conformational energy space for the protein defines the accessibility of different protein conformers. If the native state of the protein is characterized by multiple minima, with low energy barriers separating the different conformations, the lock and key model would be far from a reasonable representation. A complete flexibility on the part of the protein corresponds to an induced fit mechanism and, in that case, the protein could adapt to the shape and requirements of the ligand. When looking at the protein as an ensemble of different conformations in thermo- dynamic equilibrium, the binding pocket is observed to be flexible because it can exist in the different states that coexist in equilibrium, from which the ligands can select the most favorable conformation. Therefore, in general, the structural confor- mation of the binding site is dictated by the ligand, as it optimizes its interactions with the ensemble of conformations that the protein can assume.12 The different models of ligand recognition may, however, coexist in a single process. For example, a stability study on 16 structurally diverse proteins was used to study flexibility in the binding site.13 Binding sites appear to have regions of high structural stability and regions with low structural stability. Highly stable regions may in fact behave as a lock, while the more flexible regions may be able to undergo induced fit. The ability of ligands to bind to proteins does not depend only on the types of residues available at the contact interface or the ability of the protein or ligand to accommodate each other via an accessible conformational change.14 Other crucial elements are the solvation and desolvation processes of the interacting pair. The environment in which the interactions take place often dictates the solvation effects. Some binding pockets are enclosed in a deep, hydrophobic space within the protein, while others are more open and have greater exposure to solvent.15 The energies involved in desolvation of ligand and protein are critical determinants of binding and therefore solvation can alter the ability of the ligands to interact with the target protein. 2.3 AMINO-ACID PREFERENCE IN BINDING SITES Protein binding sites are characterized as having certain amino acids with properties that confer an ability to form binding interactions. The structural data available in protein crystallographic databases have shown that hydrophobic residues are over- utilized in the interior of proteins, and hydrophilic amino acids abound at the surfaces. It has long been known that antibodies have complementarity-determining regions with a distinct frequency of specific amino acids like Tyr and Trp.16 Despite the limited types of amino acids present, the differential usage of these residues was proposed to account for the specificity observed in antibodies. A similar finding has been described for enzymes.17,18 Large bulky amino acids, such as Trp and Tyr, and His and Arg, are overrepresented in binding sites compared to bulk protein. Even in the case of protein–protein interactions, where a small number of surface residues are responsible for most of the energy of interaction, the residues at the interface have a composition very different from that of the rest of the protein. The preference for certain residues in antibodies, enzyme binding sites, or at protein–protein interaction sites limits the number of possible binding motifs, which in turn limits the specificity that can be achieved. At the same time, it suggests that DK3714_C002.fm Page 15 Monday, February 5, 2007 12:04 PM
- 43. 16 Spectral Techniques in Proteomics certain features may repeat across different proteins, which could be useful for classification of binding sites. The fact that commonalities exist in binding sites is reinforced by the observation that a bound ligand can stabilize proteins against thermal denaturation. When screening for ligands using this property, up to 10% of the hits were found to have biologically relevant activities and consequently reflect binding at the active site or a modulating site.19 The high hit rate is clearly larger than what would be expected if the ligands were binding at random throughout the protein surface. It suggests that binding sites, as compared to other regions of the protein, have characteristics that make them hospitable to ligands. All of these studies support the idea of amino-acid preferences found in protein binding sites. The prevalence of conserved residues has been used to predict the location of binding sites without the use of structural information. Neural network algorithms using protein sequence profiles were developed to successfully identify sites of protein–protein20 and protein–DNA21 interactions. Furthermore, a study by La et al.22 showed that binding pockets in proteins could be identified from sequence informa- tion using phylogenetic motifs, which are sequence regions conserved in a protein family phylogeny.22 2.4 CONSERVED SEQUENCE AND STRUCTURAL MOTIFS AT BINDING SITES Although sequence information is certainly useful, structural characterization of binding sites affords even greater utility. Similar spatial arrangements of particular residues in different proteins have been known for some time. The most familiar example is probably that of serine proteases, which share the same catalytic triad despite having diverse folding motifs and over 60 different phylogenetic families.23 The triad of histidine, aspartate, and serine residues is arranged similarly in three- dimensional space. A fatty acid cleaving protein, lipase, is a key enzyme in the regulation of lipids and shares this same catalytic triad,24 while acetylcholinesterases have a very similar triad, with a conservative substitution of glutamate for aspartate.25 Nucleotide cofactor recognition by proteins also shows remarkable similarities despite the considerable differences in primary sequence and chain folding. The nucleotide recognition domain in glycosyltransferases26 is also found in multiple species. Recognition of adenylate by structurally diverse proteins has been shown to have a characteristic signature in overall energy calculations, despite variations in the utilization of different residues.27 From the ligand point of view, most cofactors show some significant degree of conformational similarity when bound. The con- servation was demonstrated for glutathione28 and adenylate, which show substantial similarity in the ligand conformation when bound to the protein. As it was pointed out, the conservation of bound ligand conformation is a constraint that can be used even if the binding motifs on the complementary protein active site are not obviously homologous in other proteins. The classification of proteins based on binding site characteristics does not necessarily agree with the classifications carried out by other means. Cappello et al.29 DK3714_C002.fm Page 16 Monday, February 5, 2007 12:04 PM
- 44. Similarities in Protein Binding Sites 17 showed that characterization of the adenine binding sites in terms of the possible hydrogen bond patterns can be used as a protein classification scheme. The resulting classification does not correspond to other classifications based on sequence or structure. Differing architectures of the binding site can provide for similar patterns of binding. For adenine, hydrophobic residues involved in stacking interactions with the aromatic portions of the ligand were found to be especially variable. Nevertheless, proteins with a different fold or even belonging to a different protein class can have adenine binding sites with similar properties in terms of the interface composition and hydrogen bond interaction patterns. In the case of the ubiquitous cofactor, nicotinamide adenine dinucleotide (NAD), we found that it was possible to make a connection between the sequences of proteins that utilize NAD and the NAD binding conformation.30 Our laboratory employed sequence clustering algorithms to characterize all NAD utilizing enzymes. The Swiss–Prot Database was chosen as the source for most of the sequence information related to NAD(P)-dependent oxidoreductases due to the high level of annotation. There were 4,613 enzyme sequences that utilize NAD(P) to perform their enzymatic functions. These sequences were subjected to an all-against-all sequence comparison using the basic local alignment search tool (BLAST), and the sequence identities were used to populate a similarity matrix. Divisive hierarchical cluster analysis grouped the sequences into 94 distinct sequence families. These sequence families correlated strongly with protein fold classifications whenever structural information was available for multiple members of a sequence family. Among the 94 sequence families, 53 were structurally characterized at the time. Each of the structurally characterized proteins in a sequence family correlated to a single protein fold and, remarkably, to a common bound conformation for NAD(P). Analysis of the crystal structures of oxidoreductases with bound NAD(P) cofactor revealed 16 different conformations and, in every case, a sequence family for which structural information was available corresponded to one and only one cofactor bioactive conformation. The results of this study are interesting because the protein classifications were carried out without incorporating protein structure information—that is, from sequence information alone.30 The correlation of sequence to NAD(P) bound confor- mation would suggest that protein sequences of certain gene families, when combined with the appropriate classification techniques, can be used to predict ligand binding conformation. Somehow, for the large oxidoreductase gene family, the sequences contain information about the ligand-preferred orientation and consequently the relative organization of the binding site. The method was used extensively31 to mine the complete genomes of 25 organisms representing bacteria, protists, fungi, plants, and animals, and 811 viruses, to identify and classify NAD(P)-dependent enzymes. In general, the distribution of these enzymes by oxidoreductase family was correlated to the number of different catalytic mechanisms in each family and suggests another important aspect of the studies.A better understanding of the ligands that proteins bind can provide us with insight into the potential mechanisms and functions of the enzymes. The sequence-based clustering methods group proteins according to the recognition motifs they use, even when they may have different overall fold. This is critical for modern techniques of drug discovery that rely on DK3714_C002.fm Page 17 Monday, February 5, 2007 12:04 PM
- 45. 18 Spectral Techniques in Proteomics conserved binding site features to create chemical libraries for entire families of protein targets.32 2.5 THREE-DIMENSIONAL DESCRIPTORS FOR BINDING SITE CHARACTERIZATION The use of three-dimensional pharmacophore descriptors derived from protein bind- ing sites has recently been proposed to classify proteins.33 The method relies on descriptors formed from molecular fragments that have been docked, minimized, filtered, and clustered in protein active sites. It builds upon a drug discovery tech- nique—multiple copy simultaneous search (MCSS)—used for the buildup of ligands in binding sites.34 The MCSS method is utilized to search for optimal positions and orientations of a set of functional groups. These fragments provide coordinates from their position, which in turn provide a summary of the shape, electrostatics, locations, and angles of entry into pockets of recognition sites. The descriptors can be used to correlate the active site pharmacophores with activity or function, although with a mixed degree of success. A particular concern for such a technique is how protein flexibility or minor changes in structure might influence the results. The descriptors are robust with respect to small changes in protein structure, as shown by a number of compounds that were cocrystallized in a protein. While the classification technique works well with tight protein families that have small root mean square deviations among family members, protein families having larger variations in active site structure are not classified optimally. For example, nuclear receptors give a tightly correlated group despite the variety of ligands considered, whereas in metalloproteases, their overall shape is a less useful feature in classifying members of that family.33 Nevertheless, the method is of particular interest for drug discovery because it demonstrates a correlation between binding site descriptors and biological classes, based on the characteristics of small molecules. A related method was presented in the literature35 in which an interaction finger- print was defined such that a one-dimensional binary string was used to represent three-dimensional structural binding information from a protein–ligand complex. Each fingerprint represents the “structural interaction profile” of the complex that can be used to organize, analyze, and visualize information encoded in ligand–receptor complexes.35 The method was used to analyze approximately 90 known x-ray crystal structures of protein kinase-inhibitor complexes obtained from the Protein Data Bank. The fingerprints allowed organization of the structures in terms of the simi- larities and diversity among their small-molecule binding interactions. Knowledge from the exponentially growing body of structurally characterized protein–ligand complexes will increasingly be exploited in structure-based drug design.32 These types of classifications based on the characteristics of protein–ligand interactions can be facilitated by receptor ligand databases. Relibase36 was developed as a database system particularly designed to handle protein–ligand-related problems and tasks. Features of Relibase include the detailed analysis of superimposed ligand binding sites, ligand similarity and substructure searches, and three-dimensional searches for protein–ligand and protein–protein interaction patterns. DK3714_C002.fm Page 18 Monday, February 5, 2007 12:04 PM
- 46. Similarities in Protein Binding Sites 19 2.6 EXPERIMENTAL EVIDENCE OF BINDING SIMILARITIES An alternative approach to the characterization of the properties of binding sites is to supplement computational parameters with experimental information. A large data collection comprising affinity estimates of small molecules for different proteins was generated. All measurable interaction strengths were recorded, including modest affinities that would be disregarded in most pharmacological screens. These affinity values can then be used as descriptors for the proteins and their ligands.37–39 As the database grew, patterns started to emerge in the data that suggested the existence of statistical relationships among affinity values, even when the proteins were unrelated by structure or function. In many cases, the relationships adopt a linear form, where the affinity of a compound for a given protein could be expressed as a weighted sum of its affinity for other unrelated proteins. This is a very important observation that suggests the presence of redundancies in the data that are accumulated in screening. In assembling a database of information intended for wide use in drug discovery, the redundancies should be minimized. Based on this idea, data for over 500 proteins was reduced to a significantly smaller set of less than 20 proteins that retained essentially all the information.38 The set containing the most information was chosen based on orthogonalization procedures.39 As a result, this subset of proteins, as a reference set, adequately represents all the data in the set studied to date. Because the original database included diverse proteins with few structural or functional similarities,40 it is also possible that the smaller reference panel can represent as-of-yet untested or uncharacterized proteins. It is an interesting phenomenon that a small reference panel could potentially represent most of the protein capabilities of the proteins that can be found in a proteome. Another study profiled a family of proteins using 20 kinase inhibitors, including 16 that are approved drugs or in clinical development, by analysis against a panel of 119 protein kinases.41 Specificity was found to vary widely and is not strongly correlated with chemical structure or the identity of the intended target. The results represent a systematic, small molecule–protein interaction map for clinical compounds across a large number of related proteins. There is a clear interest in exploiting the information contained in chemical databases of protein–ligand interactions and in the development of new tools centered on the use of such information. For the most part, the basic concept is simple in that compounds that share similarities in their binding to a set of proteins are expected to elicit similar pharmacological responses. The BioPrint database was constructed by systematic profiling of nearly all drugs available on the market, as well as numerous reference compounds.42,43 The database is composed of several large datasets: com- pound structures and molecular descriptors; in vitro absorption, distribution, metabo- lism, and excretion (ADME) and pharmacology profiles; and complementary clinical data including therapeutic use information, pharmacokinetics profiles, and adverse drug reaction (ADR) profiles. The platform represents a systematic effort to enhance the use and reliability of in silico methods to predict potential clinical liabilities.44 The experimental activity profiles define an “activity space” in which drugs and reference compounds are positioned in coordinates that describe inhibitory propen- sities, thereby unambiguously characterizing a molecule in terms of its receptor DK3714_C002.fm Page 19 Monday, February 5, 2007 12:04 PM
- 47. 20 Spectral Techniques in Proteomics binding properties. Even if their implementation is novel, conceptually similar approaches have been described in the literature. These approaches implicitly acknowledge the existence of similarities in binding sites and the transferability of information that can be obtained even among unrelated targets.37–43 The binding process can be simulated by computational means using docking procedures.45 The redundancy in the data is also observed in docking scores. Even in that case, the scores for the interaction between a series of proteins and a ligand can be used to represent the interaction scores of an unrelated protein for the same set of compounds. As with the experimental results, transferability of binding infor- mation is found even when there is no obvious primary or tertiary homology among the structures. Some general characteristics of the binding site such as its shape and size may limit the types of ligands that can be favorably accommodated. As such, the correlations observed could be a reflection of the types of compounds a site is unable to recognize as much as what types of compounds it actually prefers to bind. 2.7 EXPLOITING THE SIMILARITIES IN PROTEIN BINDING SITES: MODULAR APPROACHES TO DRUG DESIGN The major focus of applied work on protein–ligand interactions is for purposes of drug design. The existence of similarities in binding sites has multiple implications for those endeavors. The first is that a drug is not a key that can open only one lock. Rather, all drugs show varying degrees of interaction with a number of proteins. Successful drugs, therefore, are not ones that interact exclusively with a target of interest. Rather, they are compounds that display the highest affinity for protein targets attributed to the desired pharmacological outcome and lowest affinity to those detrimental to the desired effects. A second important lesson is that the effort to evaluate compounds against the target and the proteins related to it, based on sequence or structural family, may be misplaced. The results show that it is just as possible for the compounds to interact with completely unrelated proteins. As a practical matter, both conclusions reinforce the importance of accruing uniform datasets on compounds that bind to proteins. The resulting database can have sig- nificant utility when properly mined. The increasing evidence for similarities in binding sites suggests that an effective way to create new ligands for proteins would be to anchor a promiscuous compound in the binding site and use flanking regions to gain specificity.2 This provides the basis for modular approaches to drug design, such as the well known structure activity relationship by nuclear magnetic resonance (SAR by NMR) technique46 or the SHAPES procedures47 based on a parallel application of other spectroscopic techniques.2,10,48 The concept has been exploited in a systems-based approach for gene families that share a common cofactor.49 The basic premise is to identify a mimic for the cofactor that has drug-like properties and can be used as the central scaffold for a parallel synthesis effort. The resulting libraries contain chemicals able to bind several different members of that gene family. Many challenges have to be overcome. First, as discussed earlier, the cofactor or other common ligands do not share the same conformation for all members of the gene family. Indeed, several different confor- DK3714_C002.fm Page 20 Monday, February 5, 2007 12:04 PM
- 48. Similarities in Protein Binding Sites 21 mations are observed for the same cofactor in crystallographic studies. Once a protein family is subdivided into classes that bind the cofactor in a similar way and a mimic for the cofactor has been identified, the next challenge is to decide how to extend the scaffold into a pocket in the protein that would confer selectivity. Spectroscopic techniques such as NMR10,48 are ideally suited for this purpose. The result is a ligand that spans more than one pocket in the binding site, with thermodynamic advantages for binding. An example using oxidoreductases illustrates the approach very clearly. The existence of similarities in binding sites supports this and other modular approaches to drug discovery. 2.8 FUTURE PROSPECTS As new techniques are developed for the characterization of interactions among proteins and between proteins and small molecules, a better understanding of the individual processes is achieved. However, we need to develop more sophisticated tools to fully extract the information that these novel techniques provide, as well as to identify relationships across datasets and disciplines. The large datasets that are being compiled in genomics, structural biology, in-vitro testing, in-vivo testing, metabolism and toxicology, and clinical trials would be wasted without effective tools for mining them.32 One of the most common queries is about similarities and differences. In the realm of molecular pharmacology, the questions of which mole- cules are similar and which are not can be reduced to the types of interactions that they make with the biological system. Those interactions occur with proteins, and the fact that the same chemicals are able to interact with a variety of proteins indicates some degree of similarity among the proteins, even though the resemblance may not be obvious. A critical need in molecular pharmacology is the development of a better under- standing of protein binding similarities and the dynamic process that occurs during the recognition process.50 The arrangement of proteins and compounds into classes is a central problem in drug discovery, and the vast amount of data being accumulated requires new ways to classify proteins and ligands. The importance is not merely theoretical, but has great practical implications. Problems of selectivity are squarely in this realm. Adverse events, environmental challenges, drug–drug interactions, and toxicological risk assessment are all related to this central problem: Small molecules interact with multiple proteins in ways that are not necessarily anticipated. If we are to increase the efficiency of the drug discovery process, we need to understand how those interactions come about and apply our expanding knowledge base in drug design to reinforce desirable characteristics and avoid unwanted ones. The interface between chemistry and the proteome, commonly referred to as chemoproteomics or chemical proteomics, will continue to provide a unique per- spective of biological systems. We showed some initial tentative steps into that area, where the classification of small molecules can be done based on a proteome or the proteome classified according to its chemical preferences. Chemoproteomics is likely to continue to advance and its development will provide critical new tools to probe biological function and advance our knowledge of systems biology. DK3714_C002.fm Page 21 Monday, February 5, 2007 12:04 PM
- 49. 22 Spectral Techniques in Proteomics REFERENCES 1. Kauvar, L.M., Villar H.O., Deciphering cryptic similarities in protein binding sites. Curr. Opin. Biotechol. 9, 390, 1998. 2. Erlanson, D.A., McDowell, R.S., O’Brien, T., Fragment-based drug discovery. J. Med. Chem. 47, 3463, 2004. 3. Jacoby, E., Davies, J., Blommers, M.J., Design of small molecule libraries for NMR screening and other applications in drug discovery. Curr. Top. Med. Chem. 3, 11, 2003. 4. LaBella, F.S., Molecular basis for binding promiscuity of antagonist drugs. Biochem. Pharmacol. 42, Suppl:S1–8, 1991. 5. Lauffenburger, D.A., Linderman, J.J., Receptors: Models for Binding Trafficking and Signaling, Oxford University Press, New York, 1998. 6. Koshland, D.E., Jr., The key-lock theory and the induced fit theory. Angew Chem-Int. Ed. 33, 2375, 1995. 7. Busenlehner, L.S., Armstrong, R.N., Insights into enzyme structure and dynamics elucidated by amide H/D exchange mass spectrometry. Arch. Biochem. Biophys. 433, 34, 2005. 8. Eyles, S.J., Kaltashov, I.A., Methods to study protein dynamics and folding by mass spectrometry. Methods 34, 88, 2004. 9. Bruschweiler, R., New approaches to the dynamic interpretation and prediction of NMR relaxation data from proteins. Curr. Opin. Struct. Biol. 13, 175, 2003. 10. Pellecchia, M., Sem, D.S., Wuthrich, K., NMR in drug discovery. Natl. Rev. Drug Discovery 1, 211, 2002. 11. Straub, F.B., Formation of the secondary and tertiary structure of enzymes. Adv. Enzymol. Related Areas Molecular Biol. 26, 89, 1964. 12. Ma, B., Shatsky, M., Wolfson, H.J., Nussinov, R., Multiple diverse ligands binding at a single protein site: A matter of pre-existing populations. Protein Sci. 11, 184, 2002. 13. Petock, J.M., Torshin, I.Y., Weber, I.T., Harrison, R.W., Analysis of protein structures reveals regions of rare backbone conformation at functional sites. Proteins 53, 872, 2003. 14. Klebe, G., Bohm, H.J., Energetic and entropic factors determining binding affinity in protein–ligand complexes. J. Recept Signal Transduct Res. 17, 459, 1997. 15. Feig, M., Brooks, C.L., III, Recent advances in the development and application of implicit solvent models in biomolecule simulations. Curr. Opin. Struct. Biol. 14, 217, 2004. 16. Mian, I.S., Bradwell, A.R., Olson, A.J., Structure, function and properties of antibody binding sites. J. Molecular Biol. 217, 133, 1991. 17. Villar, H.O., Kauvar, L.M., Amino acid preferences at protein binding sites. FEBS Lett. 349, 125, 1994. 18. Villar, H.O., Koehler, R.T., Amino acid preferences of small, naturally occurring polypeptides. Biopolymers 53, 226, 2000. 19. Bowie, J.U., Pakula, A.A., Screening method for identifying ligands for target proteins. U.S. Patent 5585277, 1996. 20. Zhou, H.X., Shan, Y., Prediction of protein interaction sites from sequence profile and residue neighbor list. Proteins 44, 336, 2001. 21. Ahmad, S., Sarai, A., PSSM-based prediction of DNA binding sites in proteins. Bioinformatics 19, 33, 2005. 22. La, D., Sutch, B., Livesay, D.R., Predicting protein functional sites with phylogenetic motifs. Proteins 58, 309, 2005. DK3714_C002.fm Page 22 Monday, February 5, 2007 12:04 PM
- 50. Similarities in Protein Binding Sites 23 23. Rawlings N.D., O’Brien E., Barrett A.J., MEROPS: The protease database. Nucleic Acids Res. 30, 343, 2002. 24. Contreras, J.A., Karlsson, M., Osterlund, T., Laurell, H., Svensson, A., Holm, C., Hormone-sensitive lipase is structurally related to acetylcholinesterase, bile salt- stimulated lipase, and several fungal lipases. Building of a three-dimensional model for the catalytic domain of hormone-sensitive lipase. J. Biol. Chem. 271, 31426, 1996. 25. Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L., Silman, I., Atomic structure of acetylcholinesterase from Torpedo californica: A prototypic acetylcholine-binding protein. Science 253, 872, 1991. 26. Kapitonov, D., Yu, R.K., Conserved domains of glycosyltransferases. Glycobiology 9, 961, 1999. 27. Moodie, S.L., Mitchell, J.B., Thornton, J.M., Protein recognition of adenylate: An example of a fuzzy recognition template. J. Molecular Biol. 263, 486, 1996. 28. Koehler, R.T., Villar, H.O., Bauer, K.E., Higgins, D.L., Ligand-based protein align- ment and isozyme specificity of glutathione S-transferase inhibitors. Proteins 28, 202, 1997. 29. Cappello, V., Tramontano, A., Koch, U., Classification of proteins based on the properties of the ligand-binding site: the case of adenine-binding proteins. Proteins 47, 106, 2002. 30. Kho, R., Baker, B.L., Newman, J.V., Jack, R.M., Sem, D.S., Villar, H.O., Hansen, M.R., A path from primary protein sequence to ligand recognition. Proteins 50, 589, 2003. 31. Kho, R., Newman, J.V., Jack, R.M., Villar, H.O., Hansen, M.R., Genome-wide profile of oxidoreductases in viruses, prokaryotes, and eukaryotes. J. Proteome Res. 2, 626, 2003. 32. Mestres, J., Computational chemogenomics approaches to systematic knowledge- based drug discovery. Curr. Opin. Drug Discovery Dev. 7, 304, 2004. 33. Arnold, J.R., Burdick, K.W., Pegg, S.C., Toba, S., Lamb, M.L., Kuntz, I.D., SitePrint: Three-dimensional pharmacophore descriptors derived from protein binding sites for family-based active site analysis, classification, and drug design. J. Chem. Inf. Computer Sci. 44, 2190, 2004. 34. Caflisch, A., Miranker, A., Karplus, M., Multiple copy simultaneous search and construction of ligands in binding sites: Application to inhibitors of HIV-1 aspartic proteinase. J. Med. Chem. 36, 2142, 1993. 35. Deng, Z., Chuaqui, C., Singh, J., Structural interaction fingerprint (SIFt): A novel method for analyzing three-dimensional protein-ligand binding interactions. J. Med. Chem. 47, 337, 2004. 36. Hendlich, M., Bergner, A., Gunther, J., Klebe, G., Relibase: Design and development of a database for comprehensive analysis of protein–ligand interactions. J. Mol. Biol. 326, 607, 2003. 37. Kauvar, L.M., Higgins, D.H., Villar, H.O., Sprotsman, J.R., Engqvist Goldstein, A., Bukar R., Bauer, K.E., Dilley, H., Rocke, D.M., Predicting ligand binding to proteins by affinity fingerprinting. Chem. Biol. 2, 107, 1995. 38. Kauvar, L.M., Villar, H.O., Sportsman, J.R., Higgins, D.L., Schmidt, D.E., Protein affinity map of chemical space. J. Chromatogr. B 715, 93, 1998. 39. Dixon, S.L., Villar, H.O., Bioactive diversity and screening library selection via affinity fingerprinting. J. Chem. Inf. Computer Sci. 38, 1192, 1998. 40. Hsu, N., Cai, D., Damodaran, K., Gomez, R.F., Keck, J.G., Laborde, E., Lum, R.T., Macke, T.J., Martin, G., Schow, S.R., Simon, R.J., Villar, H.O., Wick, M.M., Beroza, P., Novel cyclooxygenase-1 inhibitors discovered using affinity fingerprints. J. Med. Chem. 47, 4875, 2004. DK3714_C002.fm Page 23 Monday, February 5, 2007 12:04 PM
- 51. 24 Spectral Techniques in Proteomics 41. Fabian, M.A., Biggs, W.H., Treiber, D.K., Atteridge, C.E., Azimioara, M.D., Benedetti, M.G., Carter, T.A., Ciceri, P., Edeen, P.T., Floyd, M., Ford, J.M., Galvin, M., Gerlach, J.L., Grotzfeld, R.M., Herrgard, S., Insko, D.E., Insko, M.A., Lai, A.G., Lelias, J.M., Mehta, S.A., Milanov, Z.V., Velasco, A.M., Wodicka, L.M., Patel, H.K., Zarrinkar, P.P., Lockhart, D.J., A small molecule–kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23, 329, 2005. 42. Horvath, D., Jeandenans, C., Neighborhood behavior of in silico structural spaces with respect to in vitro activity spaces—A novel understanding of the molecular similarity principle in the context of multiple receptor binding profiles. J. Chem. Inf. Computer Sci. 43, 680, 2003. 43. Krejsa, C.M., Horvath, D., Rogalski, S.L., Penzotti, J.E., Mao, B., Barbosa, F., Migeon, J.C., Predicting ADME properties and side effects: The BioPrint approach. Curr. Opin. Drug Discovery Dev. 6, 470, 2003. 44. Engelberg, A., Iconix Pharmaceuticals, Inc.—Removing barriers to efficient drug discovery through chemogenomics. Pharmacogenomics 5, 741, 2004. 45. Koehler, R.T.,Villar, H.O., Statistical relationships among docking scores for different protein binding sites. J. Computer Aided Molecular Design 14, 23, 2000. 46. Shuker, S.B., Hajduk, P.J., Meadows, R.P., Fesik, S.W., Discovering high-affinity ligands for proteins: SAR by NMR. Science 274, 1531, 1996. 47. Fejzo, J., Lepre, C.A., Peng, J.W., Bemis, G.W., Ajay, Murcko, M.A., Moore, J.M., The SHAPES strategy:An NMR-based approach for lead generation in drug discovery. Chem Biol. 6, 755, 1999. 48. Villar, H.O., Yan, J., Hansen, M.R., Using NMR for ligand discovery and optimiza- tion. Curr. Opin. Chem. Biol. 8, 387, 2004. 49. Sem, D.S., Bertolaet, B., Baker, B., Chang, E., Costache, A.D., Coutts, S., Dong, Q., Hansen, M., Hong, V., Huang, X., Jack, R., Kho, R., Lang, H., Ma, C., Meininger, D., Pellecchia, M., Pierre, F., Villar, H.O., Yu L., Systems-based design of bi-ligand inhibitors of oxidoreductases: Filling the chemical proteomic toolbox. Chem. Biol. 11, 185, 2004. 50. Cavasotto, C.N., Abagyan, R.A., Protein flexibility in ligand docking and virtual screening to protein kinases. J. Molecular Biol. 337, 209, 2004. DK3714_C002.fm Page 24 Monday, February 5, 2007 12:04 PM
- 52. 25 3 Survey of Spectral Techniques Used to Study Proteins Daniel S. Sem CONTENTS 3.1 Introduction.................................................................................................... 26 3.2 Mass Spectrometry......................................................................................... 26 3.2.1 Background and History.................................................................... 26 3.2.2 Ionization Method.............................................................................. 26 3.2.2.1 MALDI ............................................................................... 27 3.2.2.2 ESI ...................................................................................... 28 3.2.3 Mass Analyzers .................................................................................. 29 3.2.3.1 TOF..................................................................................... 29 3.2.3.2 Quadrupole.......................................................................... 29 3.2.3.3 Ion Trap............................................................................... 29 3.2.3.4 FT-ICR ................................................................................ 30 3.2.4 Tandem MS........................................................................................ 30 3.3 Spectroscopic Techniques.............................................................................. 30 3.3.1 Background and Survey..................................................................... 30 3.3.2 UV-Visible.......................................................................................... 31 3.3.3 Fluorescence....................................................................................... 32 3.3.4 Magnetic Resonance .......................................................................... 35 3.3.4.1 NMR ................................................................................... 35 3.3.4.2 EPR ..................................................................................... 36 3.3.5 IR and Raman .................................................................................... 37 3.3.6 SPR..................................................................................................... 38 3.3.7 X-Ray Crystallography ...................................................................... 39 3.4 Future Prospects............................................................................................. 40 Acknowledgments.................................................................................................... 41 References................................................................................................................ 41 DK3714_C003.fm Page 25 Monday, February 5, 2007 10:55 AM
- 53. 26 Spectral Techniques in Proteomics 3.1 INTRODUCTION Proteins can be studied with a wide range of spectral techniques, of which only a small subset are widely use in proteomics. Protein spectral techniques, for the purposes of this book, are categorized as: • Mass spectrometry (MS) techniques • Spectroscopic techniques Mass spectrometry involves the measurement of protein mass-to-charge (m/z) ratios, from which molecular weights of intact proteins and their fragments can be calculated. Spectroscopic techniques all involve monitoring the interaction of electro- magnetic radiation with matter (table 3.1). Since there are far too many mass spectrometry and spectroscopic techniques to discuss in a chapter as short as this, even in a cursory manner, emphasis will be placed on those currently being applied in proteomics. For more detailed discussion of these methods, the reader is referred to some of the many excellent books and articles that served as primary sources for this chapter [1–9]. 3.2 MASS SPECTROMETRY [3] 3.2.1 BACKGROUND AND HISTORY Mass spectrometry was applied to small molecules long before it was used to study proteins. The first mass spectrometer can be dated to 1912 (J. J. Thompson), with the first atomic weight measurement made in 1919. The 1950s saw the development of quadrupole analyzers and the first gas chromatography (GC)-MS. The 1960s saw the development of tandem MS and electrospray ionization (ESI), with subsequent devel- opment of liquid chromatography (LC)-MS in 1973 (McLafferty). But MS did not find broad use for protein studies until the 1980s—first with the development of FAB (fast atom bombardment) MS in 1981 and then application of ESI to macromolecules in 1984; these were followed by development of ion cyclotron resonance MS. These developments opened the door to the application of MS in proteomics in the 1990s, when protein sequencing with matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI MS/MS) was introduced. Today, MS has evolved to be the most prominent spectral technique used in proteomic studies. Many technological developments have been and continue to be made, at an exciting pace. This chapter and this book attempt only to provide a snapshot in time of some of the more widely used MS techniques and make no attempt to provide a comprehensive overview of the field. A general description is provided for the more commonly used mass spectrometers in terms of ionization method and mass analyzers, since these are the components of greatest variability and importance in the purchase of an instrument. 3.2.2 IONIZATION METHOD Proteins must be introduced into the mass spectrometer and ionized so that they can travel through the mass analyzer and to the detector (fig. 3.1). In the early days of DK3714_C003.fm Page 26 Monday, February 5, 2007 10:55 AM
- 54. Survey of Spectral Techniques Used to Study Proteins 27 mass spectrometry, EI (electron ionization) was the method of choice for ionization, but this would fragment the molecule. Such fragmentation provides useful structural information for small molecules, but makes spectra of proteins overly complex and uninterpretable. For this reason, softer ionization methods were developed. FAB was developed first, but had a limited molecular weight range (<7000 g/mol). Later, MALDI and ESI were developed for protein applications, and these are currently the most widely used ionization techniques. 3.2.2.1 MALDI MALDI is described in detail in chapter 5, and elsewhere in this book. In MALDI, the protein sample is mixed with a solid “matrix” material, then introduced into the TABLE 3.1 Spectroscopic Techniques Used to Study Proteins Technique Measured (most common) Electronic transitions Fluorescence,a including FPa (fluorescence polarization) and FRETa (fluorescence resonance energy transfer) Binding; structure; dynamics UV-visible (UV-vis) absorbance spectroscopy Electronic structure; binding CD (circular dichroism) and MCD (magnetic circular dichroism) Secondary structure; bonding Vibrational transitions IRa (infrared) Structure; bonding Raman,a resonance Raman,a and polarized Raman Structure; bonding VCD (vibrational CD) Structure; bonding Electron and/or nuclear spin transitions NMRa (nuclear magnetic resonance) Structure and dynamics CIDNP (chemically induced dynamic nuclear polarization) Structure EPRb (electron paramagnetic resonance) Structure (paramagnetic) ENDOR (electron nuclear double resonance) Structure (paramagnetic) and bonding ESEEM (electron spin-echo envelope modulation) Structure (paramagnetic) and bonding Other SPRa (surface plasmon resonance) Binding X-ray crystallographya Structure SLS/DLS (static light scattering/dynamic light scattering) Size (mol. wt.; aggregation) SAXS (small- [or low]-angle x-ray scattering) Size (mol. wt.; aggregation) Mössbauer and magnetic Mössbauer Bonding (metal) XAFS/EXAFS (x-ray absorption fine structure/extended x-ray absorption fine structure) Bonding (metal) XANES (x-ray absorption near-edge structure) Bonding (metal) XAS (x-ray absorption spectroscopy) Bonding (metal) a Techniques discussed in this chapter and elsewhere in this book. b Also referred to as ESR (electron spin resonance). DK3714_C003.fm Page 27 Monday, February 5, 2007 10:55 AM
- 55. Other documents randomly have different content
- 56. They uttered a shout of joy on perceiving their chiefs, and, eagerly rising, ran to meet them. "Good day, gentlemen," Leon said, as he leaped from his horse. "I am rather behind my time, but you must blame the night storm, which compelled us to halt on the road. Is there any news?" "None, captain," they answered. "In that case listen to me. Ten of you will stay here, and at four o'clock tomorrow morning proceed with twelve mules to the house of Don Juan y Soto-Mayor, and place yourselves at the orders of that gentleman, whom you will accompany to Valdivia." Diego set about selecting the men whom he thought the best fitted for the expedition; and after he had done so, Leon addressed the others. "You will start for Valparaíso and await my orders there; you will lodge at Crevel's, in the Calle San Agostino, and at Dominique the Italian's, at the Almendral. Above all," he added, "be prudent, and do not attract attention; amuse yourselves like good fellows, but do not quarrel with the señores, or have any fights with the sailors. You understand me, I suppose?" "Yes, captain," they all answered. "Very well. Now I will give each of you five ounces to cover your expenses, and do not forget that I may want you at any moment, and you must be ever ready to obey my summons." He gave them the money, and after repeating his recommendations, he retired, leaving it to Diego to give the men who were proceeding to Valparaíso the final instructions which they might need. The smugglers removed all traces of their meal, and each of them hurried to saddle his horse. A few minutes later, forty men of the band set out under the guidance of the oldest among them. Diego watched them start, and then returned to Leon, who was resting from his fatigue on a small turf mound, overshadowed by a magnificent clump of trees. The Vaquero held in his hand the alforjas which he had taken off his horse; he examined the place
- 57. where Leon was seated, and finding it as he wished, he sat down by his side; then taking out of the bag a clumsy carved earthern pipe, into which he fitted a long stem, he began to strike a light over a small horn box filled with burnt rags, which soon caught fire. When his pipe was lighted, he began smoking silently. Leon, on seeing these preparations, understood that something important was about to take place between him and Diego, and waited. At the expiration of five minutes, the latter passed him his pipe; Leon drew several puffs and then returned it to him. These preliminaries completed, Diego began to speak. "Leon, three years have passed since Heaven brought us together on the pampas of Buenos Aires; since that moment—and I shall never forget it, brother—everything has been in common between us—pleasure and pain, joy and sorrow." Leon bowed his head in the affirmative, and the half-breed continued: "Still, there is one point upon which our mouths have ever remained silent, and it is the one which refers to the life of each of us before that which we now lead together." Leon looked at him in amazement. "It is not a want of confidence," Diego hastily added, "but the slight interest we felt in cross-questioning each other, which alone is the cause. Of what use is it to know the past life of a man, if from the day when you first saw him he has not ceased to be honest and loyal? Besides, the hours are too short in the pampas for men to dream of asking such questions." "What are you coming to?" Leon at length asked. "Listen, brother. I will not question you about what I care little to know, but I wish to tell you something you must know. The moment has arrived to speak; and though the story I have to tell you is gloomy and terrible, I am accomplishing a duty." "Speak, then," said Leon.
- 58. The half-breed passed his hand over his forehead, and for a moment collected his recollections. Leon waited in silence. CHAPTER V. THE INCA OF THE NINETEENTH CENTURY. "Long ago, very long ago," Diego, the Vaquero, began, "all the lands bordering the bay of Valparaíso belonged to the Indians, whose vast hunting grounds extended on one side from the lofty peaks of the Cordilleras down to the sea, and on the other covered the Pampas of Buenos Aires, of Paraguay—in a word, all the splendid countries from which they have eternally disappeared, and it is impossible to find a trace of the moccasins which trod them during centuries." "The Indians were at that day free, happy, powerful, and more numerous than the grains of sand in the bed of the sea. But one day strange news spread among them: it was said that white men, who had come no one knew whence, and mounted on immense winged horses, had suddenly appeared in Peru." "I need not remind you of all that occurred in consequence of this news, which was only too true, or describe to you the hideous massacres committed by the Spaniards, in order to reduce the unhappy Indians to slavery, for it is a story which everybody knows. But what you are possibly ignorant of is, that during one of the dark and stormy nights which followed this invasion, a dozen men of majestic demeanour, with haughty though care-laden brows, were seen to land from a canoe half broken by the waves and jagged rocks." "They were Indians who had miraculously escaped from the sack of Quito, and had come to present themselves as suppliants to the
- 59. elders of the Araucano nation. Among them was a man whom they respectfully obeyed. He was the son of the sister of the valiant Atahualpa, King of Quito, and his name was Tahi-Mari. When in the presence of the elders, Tahi-Mari gave them a narration of the misfortunes which had struck him." "He had a daughter, Mikaa, the purest and loveliest of the daughters of the Sun. When conquered by the Spaniards, who, after killing two of his sons, set fire to his palace, Tahi-Mari, followed by his three sons left home, rushed toward the palace of the Sun, in order to save his daughter, if there were still time." "It was night: the volcano was roaring hoarsely, and hurling into the air long jets of fire, whose lurid and sinister gleams combined with the flames of the fire kindled by the conquerors of this unhappy city. The squares and streets were encumbered with a terrified multitude, who fled in all directions with terrible cries from the pursuit of the Spanish soldiers, who, intoxicated with blood and carnage, massacred mercilessly old men, women, and children, in order to tear from their quivering bodies the gold collars and ornaments which they wore. Neither tears, prayers, nor entreaties succeeded in moving their ferocious executioners, who with yells and shrill whistles excited their dogs to help them in this horrible manhunt." "When Tahi-Mari reached the Temple of the Sun, that magnificent edifice, which contained such riches, had become a prey to the flames; a girdle of fire surrounded it on all sides, and from the interior could be heard the groans of the hapless virgins who were expiring in the tortures of a horrible death. Without calculating the imminency of the peril, the poor father mad with grief and despair, rushed into the burning furnace which opened its yawning mouth before him." "'My daughter! my daughter!' he cried. In vain did the flames singe his clothing; in vain did frightful burns devour his hands and face: he felt nothing, saw nothing; from his panting chest constantly issued the piercing cry—"
- 60. "'My daughter! my daughter!'" "Suddenly a half-naked virgin, with dishevelled hair, and her features frightfully contracted, escaped from the flames; it was Mikaa. Tahi- Mari, forgetting all that he had suffered, weepingly opened his arms to the maiden, when a Spaniard, dressed in a brilliant garb, and holding a sword in his hand, rushed upon Mikaa, and ere her father had time to make a gesture thrust his weapon into her chest!" "Oh, it is frightful!" Leon, who had hitherto listened to his comrade's story in silence, could not refrain from exclaiming. Diego made no reply, but a sinister smile played round his livid lips. "The maiden fell bathed in her blood, and Tahi-Mari was about to avenge her, when the Spaniard dealt him such a fierce blow that he lost his consciousness. When he regained his senses the officer had disappeared." "It is infamous," Leon said again. "And that officer's name was Don Ruíz de Soto-Mayor," Diego said, in a hollow voice. "Oh!" Leon muttered. "Wait a moment, brother; let us continue, for I have not finished yet." "Though tracked like a wild beast, and incessantly hunted by the Spaniards, Tahi-Mari, accompanied by his three sons and some faithful friends, succeeded in getting away from Quito and reaching the country of the Araucanos." "After the Inca had recounted his misfortunes to the great Indian Chief, the latter welcomed the fugitives with hearty marks of affection; one of them, the venerable Kouni-hous-koui (he who is respected), a descendant of one of the oldest families of the Sagamores of the nation, exchanging his calumet with Tahi-Mari, declared to him, in the name of the Araucanos, that the Council of Elders adopted him as one of their caciques."
- 61. "From this day Tahi-Mari, owing to his courage and wisdom, acquired the esteem of those who had given him a new country to love and defend." "Several years passed thus, and no sign led the Araucanos to suspect that the Spaniards would ever dare to attack them; they lived in a perfect state of security, when suddenly and without any justification for the aggression, a Spanish fleet consisting of more than thirty brigantines sailed into the bay of Valparaíso. They had no sooner disembarked than they built a city, which soon saw the flag of conquest floating from its walls." "Still the Araucanos, although driven back by their terrible enemies, were aroused by the voice of Tahi-Mari, and resolved to keep the Spaniards constantly on their defence, by carrying on against them a war of snares and ambushes, in which the enemy, owing to their ignorance of the places where they fought, did not always get the best of it." "In the course of time, this perpetual war made them lose a great number of soldiers, and feeling desperate at seeing several of their men fall daily under the blows of invisible enemies, who seemed to inhabit hollow trees, the tops of mountains, or the entrails of the earth, they turned all their rage against Tahi-Mari, whose influence over all the men who surrounded him they were aware of, and resolved to get hold of him." "But it was no easy matter, for the Inca was on his guard against every attack, and was too well versed in the tactics of his enemy to let himself be caught by cunning or treachery. And yet this was destined to happen. There was among the Indian prisoners—alas! it is disgraceful to say it, but it was so—a man who, given to habits of intoxication and brought to Peru by the Spaniards, did not recoil before the offer made him to betray his brothers, on condition that they should give him as much aguardiente as he could drink." "The Spanish captain, fertile in expedients, who had proposed this cowardly bargain to the Indian, induced the latter to go to Tahi-Mari,
- 62. give himself out as an escaped prisoner, and, after inquiring into his plans, urge him to surprise the Spaniards, of whose numbers, position, and plan of campaign he was to give a false account. Once that Tahi-Mari was in the power of the Spaniards, firewater would amply compensate the traitor." "All was carried out in the way the officer suggested; for could Tahi- Mari suspect that an Araucano would betray him? He received him on his arrival among his brothers with transports of joy, and then questioned him as to the enemy's strength and means of defence. This was what the Indian was waiting for: he answered the questions asked him by adroitly dissimulating the truth, and ended by asserting that nothing was easier than to take the Spanish troops prisoners, and he offered to guide the expedition in person." "The hope of a certain victory animated the Araucanos, who joyfully greeted this proposition, and all was soon arranged for the start. During the night following the traitor's arrival, five hundred men picked from the bravest, and led by Tahi-Mari, descended the mountain under the guidance of the treacherous Indian, and marched silently upon a Spanish redoubt, in which they expected to find the principal chiefs of the enemy and surprise them." "But as they advanced they perceived a dark line which was almost blended with the darkness, but which could not escape the piercing glances of the Indians. This line formed an immense circle, which surrounded them and became more contracted every moment. It was the Spanish horse coming to meet them and preparing to attack them." "All at once Tahi-Mari uttered a yell of fury, and the head of the traitor who had drawn them into the snare rolled at his feet; but ere the Araucanos had time to retire, a number of horsemen, holding in leash twenty of those ferocious dogs trained for man hunting, rushed upon them. They were compelled to fight, and a terrible massacre began, which lasted all night. Tahi-Mari performed prodigies of valour. In the height of the action his eyes were injected with blood and a lurid pallor covered his face; he had recognised
- 63. among those who were fighting the Spanish officer who killed his daughter Mikaa on the threshold of the Temple of the Sun in so dastardly a way. On his side the Spaniard rushed with incredible fury upon the Inca." "It was a sublime moment! The two men attacked each other with equal fury, and the blood that flowed from their wounds stained their weapons. The axe which the Inca held was already whirling above the head of the Spaniard to deal him the final blow, when Tahi-Mari fell back, uttering a yell of pain: an enormous hound coming to the officer's assistance, had ripped open the Inca's stomach. Taking advantage of Tahi-Mari's defenceless state, Don Ruíz de Soto-Mayor despatched him by passing his sword right through his body." "The next day the Inca's body, frightfully mutilated, was burnt on the public square of Valdivia, in the presence of a few Indians, who had only escaped the sword of their murderers to die at a later date in the punishment of a horrible captivity." "Oh!" Leon exclaimed, who had felt his heart quiver; "it is frightful!" "What shall I say, then?" Diego asked in his turn; "I who am the last of the descendants of Tahi-Mari!" At this unexpected revelation Leon started; he looked at Diego, and understood that there was in this man's heart a hatred so deeply rooted, and, above all, so long repressed, that on the day when it broke out no power in the world would be strong enough to check the terrible effects of its explosion. He hung his head, for he knew not what to reply to this man who had to avenge such blood-stained recollections. Diego took his friend's hand, and remarking the emotion he had produced, added— "I have told you, brother, what the ancestors of Don Juan de Souza y Soto-Mayor made mine suffer, and your heart has bounded with indignation, because you are loyal and brave; but what you do not yet know is that the descendants of that family have faithfully followed the conduct of the murderers of Tahi-Mari. Oh! there are
- 64. strange fatalities in a man's life! One day—and that day is close at hand—you shall know the details of the existence which I have led, and the sufferings which I have endured without a murmur; but at the present day I will only speak of those of my race; afterwards I will speak of myself." While uttering the last words, a flash of joy like that which a tiger feels when it holds a quivering prey under its claws passed into the half-breed's eyes. He continued— "My father died a victim to the cruelty of the Spaniards, who put him to death because he dreamed of the independence of his country; his brother followed him to the tomb, weeping for his loss." "Diego! God has cruelly tried thee." "I had a mother," Diego went on, with a slight tremor in his voice; "she was the object of my father's dearest affections, and was young and lovely. One day when she left the mountain to visit my father, who was expiating within the walls of Valparaíso prison his participation in a movement which had broken out among the Araucanos, she met on the road a brilliant Spanish cavalier who wore a lieutenant's epaulettes." "The Spaniard fixed upon her an impassioned glance; she was alarmed, and tried to fly, but the horseman prevented her, and in spite of her prayers and supplications, she could not liberate herself from the villain's arms. On the morrow Lieutenant Don Juan de Soto- Mayor was able to boast among his friends, the noble chiefs of the Spanish army, that he had possessed the chaste wife of Tahi-Mari the Indian." "Yes, it was again a Soto-Mayor. This accursed name has ever hovered over the head of each member of my family, to crush it under punishment, sorrow, shame, or humiliation. Each time that one of us has reddened American soil with his blood, it was a Soto- Mayor that shed it. Each time that a member of this family met a member of mine, one was the executioner, the other the victim."
- 65. "And now, brother, you will ask me why, knowing that General Don Juan de Souza y Soto-Mayor is the man who dishonoured my mother, I did not choose among the weapons which hung from my girdle the one which should pierce his heart?—why I have not some night, when all were sleeping at the hacienda, carried within its walls the all-devouring fire, and taken, according to Indian custom, eye for eye and tooth for tooth?" "Yes, I confess it; I should have quivered with pleasure had I seen all the Soto-Mayors, who live calm and happy a few leagues from us, writhing in the agonies of death. But I am the son of Tahi-Mari, and I have another cause to defend beside my own—that of my nation. And on the day when my arm falls on those whom I execrate, it will not be the Soto-Mayors alone who perish, but all the Spaniards who inhabit these countries." "Ah! is it not strange to dream of enfranchisement after three hundred years of slavery? Well, brother, the supreme moment is close at hand; the blood of the Spaniard will again inundate the soil of Peru, and the nineteenth century will avenge the sixteenth." "That is the reason why you saw me so silent at the general's house; that is why I agreed to escort him and his family to Valdivia, for my plans are marvellously served by this journey. As for the girl you love, as I told you, you shall see her again, and it will be the beginning of the punishment which is destined to fall on this family." Diego had risen, but a moment later he resumed his ordinary stoicism. "I have told you what you ought to know, in order to understand and excuse what you may see me undertake against the Spaniards; but before going further it is right that I should know if I can count on your help, and if I shall find in you the faithful and devoted friend who never failed me up to this day." A violent contest was going on in Leon's heart. He asked himself whether he, who had no cause of complaint against the Spaniards, had any right to join those who were meditating their ruin. On the
- 66. other hand, the sincere friendship which he felt for the Vaquero, whose life he had shared during the last four years, rendered it a duty to assist him, and did not permit him to abandon him in the moment of danger. Still he hesitated, for a secret anxiety kept him undecided, and prevented him forming a resolution. "Diego," he asked the Vaquero in his turn, "before answering you, let me ask you one question?" "Speak, brother!" Diego answered. "What do you mean to do with Doña Maria?" "I have promised you to bring her to your knees. If she love you, she will be my sister; if she refuse your love, I shall have the right to dispose of her." "And she will have nothing to fear till I have seen her again?" Leon asked further. "Nothing! I swear to you." "In that case," said Leon, "I will take part in your enterprise. Your success shall be mine, and whatever be the road you follow, or the means you employ to gain the object of your designs, I will do all that you do." "Thanks, brother; I was well aware that you would support me in the struggle, for it is in the cause of justice. Now I will set out." "Do you go alone?" "Yes, I must." "When shall I see you again?" "Tomorrow morning, at Don Juan's, unless I am compelled to remain at the place where I am going longer than I think; in that case I will join you on the Talca road. Besides, you do not require me to escort the general: our men will be at their post tomorrow, and you can say something about my going on ahead." "That is true; but Doña Maria?"
- 67. "You will see her again soon. But start alone tomorrow for the country house, and I will meet you this day week, whatever may happen, in the Del Solar wood, at the San Francisco Solano quarry, where you will order a halt." "Agreed, and I leave you to act as you think proper. Next Wednesday at the Del Solar wood, and if you wish to join us before then, we shall follow the ordinary road." "Very good; now I am off." Ten minutes after this long interview, Diego was galloping away from his comrade, who watched him depart, while striving to conjecture in what direction he was going. Profoundly affected by the varied events of the preceding day, and the story which Diego had told him, Leon reflected deeply as he walked toward the smugglers remaining with him, and who were engaged in getting their weapons in order. Although nothing in his exterior announced the preoccupation from which the was suffering, it could be guessed that he was in a state of lively anxiety. The image of Doña Maria floated before his eyes; he saw her pale and trembling after he had saved her from his horse's rush, and then, carrying himself mentally within the walls of the convent of the Purísima Concepción, he thought of the barrier which separated them. Then suddenly the half-breed's words returned to his ear—"If she refuse your love," he had said, "I shall have the right to dispose of her!" An involuntary terror seized on the young man at this recollection. In fact, was it presumable that Doña Maria loved him? and would not the Vaquero be compelled to employ violence in carrying out his promise of bringing him into the presence of the novice? In that case, how could he hope to make himself loved? These reflections painfully agitated Leon Delbès, who, obeying that spontaneity of action peculiar to his quick and impetuous character, resolved to fix his uncertainty by assuring himself of the impression
- 68. which he had produced on the heart of the maiden, whom he loved with all the strength and energy of a real passion. Such a sudden birth of love would appear strange in northern countries, where this exquisite feeling is only developed in conformity with the claims of the laws of civilization; but in Chili, as in the whole of South America, love, ardent as the fires of the sun which illumines it, bursts forth suddenly and displays itself in its full power. The look of a Chilian girl is the flush which enkindles hearts of fire which beat in breasts of iron. Leon was a Frenchman, but several years' residence in these parts, and his complete adoption of American manners, customs, and usages had so metamorphosed him, that gradually his tastes, habits, and wants had become identified with those of the inhabitants of Chili, whom he regarded as his brothers and countrymen. Without further delay, then, Leon prepared to return to Valparaíso, and make inquiries about Doña Maria. "It is two o'clock," he said to himself, after consulting his watch; "I have time to ride to Ciudad, set Crevel to work, and be at the general's by the appointed hour." And leaping on his horse, he galloped off in the direction of the Port, after bidding the ten men of the escort to start with or without him the next morning for the country house. CHAPTER VI. THE BANIAN'S HOUSE. Valparaíso, like nearly all the commercial centres of South America, is a collection of shapeless huts and magnificent palaces, standing side by side and hanging in long clusters from the sided of the three
- 69. mountains which command the town. The streets are narrow, dirty, and almost deprived of air, for the houses, as in all American towns, have a tendency to approach each other, and at a certain height form a projection of four, or even six feet over the street. Paving is perfectly unknown; and the consequence is, that in winter, when the deluging rains, which fall for three months almost without leaving off, have saturated the ground, these streets become veritable sewers, in which pedestrians sink up to the knee. This renders the use of a horse indispensable. Putrid and pestilential miasmas exhale from these gutters, which are filled with rubbish of every description, resulting from the daily sweepings of the houses. On the other hand, the squares are large, square, perfectly airy, and lined with wide verandahs, which at midday offer a healthy protection from the sun. These verandahs contain handsome shops, in which the dealers have collected, at great cost, all that can tempt purchasers. It is a medley of the most discordant shops and booths, grouped side by side. A magnificent jeweller displays behind his window diamond necklaces, silver spurs, weighing from fifteen to twenty marcs, rings, bracelets, &c.; between a modest grocer quarrelling with his customers about the weight, and the seller of massamorra broth, who, with sleeves tucked up to the elbow, is selling his stuff by spoonfuls to every scamp who has an ochavo to regale himself with. The smuggler captain passed gloomily and thoughtfully through the joyous population, whose bursts of laughter echoed far and wide, and whose merry songs escaped in gay zambacuecas from all the spirit shops which are so frequent at Valparaíso. In this way he reached Señor Crevel's inn, who uttered a cry of joy on perceiving the captain, and ran out to hold his horse. "Are my men here?" Leon asked civilly, as he dismounted. "They arrived nearly two hours back," Crevel answered, respectfully. "It is well. Is the green chamber empty?"
- 70. Every landlord, in whatever country he may hang out his sign, possesses a separate room adorned with the names of blue, red, or green, and which he lets at a fabulous price, under the excuse that it is far superior to all the others in the house. Señor Crevel knew his trade too well not to have adopted this habit common to all his brethren; but he had given the name of the green room to a charming little quiet nook, which only his regular customers entered. Now, as we have said, the smugglers were very old friends of Crevel. The door of the green room, perfectly concealed in the wall, did not allow its existence to be suspected; and it was in this room that the bold plans of the landlord's mysterious trade, whose profits were far greater than those which he drew from his avowed trade, were elaborated. On hearing Leon's question, the Banian's face assumed an expression even more joyous than that with which he had greeted the young man's arrival, for he scented, in the simple question asked him, a meeting of smugglers and the settlement of some affairs in which he would have his share as usual. Hence he replied by an intelligent nod, and added aloud, "Yes, señor; it is ready for your reception." After handing the traveller's horse to a greasy waiter, whom he ordered to take the greatest care of it, he led Leon into the interior of the inn. We are bound to confess that if the architect who undertook to build this house had been more than saving in the distribution of ornamentation, it was admirably adapted for its owner's trade. It was a cottage built of pebbles and beams, which it had in common with the greater portion of the houses in Valparaíso. Its front looked, as we know, upon the Calle San Agostino, while the opposite side faced the sea, over which it jutted out on piles for some distance. An enormous advantage for the worthy landlord, who frequently profited by dark or stormy nights to avoid payment of customs dues, by receiving through the windows the goods which the smugglers sold him; and it also favoured the expeditions of the
- 71. latter, by serving as a depôt for the bales which they undertook to bring in on account of people who dealt with them. This vicinity of the sea also enabled the Frenchman, whose customers were a strange medley of all sorts of men, not to trouble himself about the result of the frequent quarrels which took place at his house, and which might have caused an unpleasantness with the police, who at Valparaíso, as in other places where this estimable institution is in vogue, sometimes found it necessary to make an example. Hence, so soon as the squadron of lanceros was signalled in the distance, Señor Crevel at once warned his guests; so that when the soldiers arrived, and fancied they were about to make a good haul, they found that the birds had flown. We need scarce say that they had simply escaped through the back window into a boat always kept fastened in case of need to a ring in the wooden platform, which served as a landing stage to the house. The lanceros did not understand this sudden disappearance, and went off with a hangdog air. Differing from European houses, which fall back in proportion to their elevation from the ground, Señor Crevel's establishment bulged outwards, so that the top was spacious and well lighted, while the ground floor rooms were narrow and dark. The landlord had always taken advantage of this architectural arrangement by having a room made on the second floor, which was reached by a turning staircase, and a perfect ear of Dionysius, as all external sounds reached the inmates, while the noise they made either in fighting or talking was deadened. The result of this was that a man might be most easily killed in the green room without a soul suspecting it. It was into this room, then, witness of so many secret councils, that the landlord introduced, with the greatest ceremony, the captain of the smugglers, who walked behind him. On regarding the interior of the room, nothing indicated the origin of its name; for it was entirely hung with red damask. Had this succeeded a green hanging? This seems to be a more probable explanation.
- 72. It received light from above, by means of a large skylight. The walls were hung with pictures in equivocal taste, representing subjects passably erotic and even slightly obscene. A large four-post bed, adorned with its tester, occupied all one side of the room, and a mahogany chest of drawers stood facing it: in a corner was a small table covered with the indispensable toilette articles—combs, brushes, &c. A small looking glass over the table, chairs surrounding a large round table, and, lastly, an alabaster clock, which for the last ten years had invariably marked the same hour between its two flower vases, completed the furniture of this famous green room. We must also mention a bell, whose string hung behind the landlord's bar, and was useful to give an alarm under the circumstances to which we have referred. Leon paid no attention to these objects, which had long been familiar to him. "Now, then," he said, as he took off his hat and poncho, and threw himself into an easy chair, "bring me some dinner at once." "What would you like, captain?" "The first thing ready: some puchero, some pepperpot—in short, whatever you please, provided it be at once, as I am in a hurry." "What will you drink?" "Wine, confound it! and try to find some that is good." "All right." "Decamp then, and make haste to bring me all I require." "Directly, captain." And Señor Crevel withdrew to attend to the preparation of the young man's dinner. During this time Leon walked up and down the room, and seemed to be arranging in his head the details of some plan he was meditating. Crevel soon returned to lay the table, which he performed without opening his lips for fear of attracting some disagreeable remark from the captain, who, for his part, did not appear at all disposed for
- 73. conversation. In an instant all was arranged with that coquettish symmetry which belongs to the French alone. "Dinner is ready, captain," said Crevel, when he re-entered the room. "Very well. Leave me; when I want you I will call you." The landlord went out. Leon sat down to the table, and drawing the knife which he wore in his boot, vigorously attacked the appetizing dishes placed before him. It is a fact worthy of remark, that with great and energetic natures, moral sufferings have scarce any influence over physical wants. It might be said that they understand the necessity of renewing or redoubling their strength, in order to resist more easily and more victoriously the griefs which oppress them, and they require all their vigour to contend worthily against them. Chilian meals in no way resemble ours. Among us people drink while eating, in order to facilitate the absorption and digestion of the food; but in America it is quite different—there people eat without drinking. It is only when the pastry and sweets have been eaten that they drink a large glass of water for digestion; then comes the wines and liqueurs, always in small quantities, for the inhabitants of hot countries are generally very sober, and not addicted to the interminable sittings round a table covered with bottles, in an atmosphere impregnated with the steam of dishes. When the meal was ended, Leon took his tobacco pouch from his pocket and rolled a cigarette, after wiping his fingers on the cloth. As this action may appear improper to the reader, it is as well that he should know that all Americans do so without scruple, as the use of the napkin is entirely unknown. Another custom worth mentioning is that of employing the fingers in lieu of a fork. This is the process among the Americans. They cut a piece of bread crumb, which they hold in their hand, and pick up with it the articles on their plate with great rapidity and cleanliness.
- 74. Nor must it be thought that they act in this way through ignorance of the fork; they are perfectly well acquainted with that utensil, and can manage it as well as we do when required; but though it is present on every table, both rich and poor regard it as an object of luxury, and say that it is far more convenient to do without it, and remark that the food has considerably more flavour when eaten in this fashion. Leon lit his cigarette, and fell again into his reflections. All at once he rose and rang the bell, and Crevel at once appeared. "Take all this away," said Leon, pointing to the table. The landlord removed all traces of the meal. "And now bring me the articles to make a glass of punch." Crevel gazed for a moment in amazement at the man who had given this order. The sobriety of the smuggler was proverbial at Valparaíso; he had never been seen to drink more than one or two glasses of Pisco, and then it was only on great occasions, or to please his friend Diego, whom he knew to be very fond of strong liquors, like all the Indians. When a bottle of aguardiente was served to the two men, the Indian finished it alone, for Leon scarce wet his lips. Hence the landlord was almost knocked off his feet on receiving his guest's unusual order. "Well, did you not hear me?" Leon resumed, impatiently. "Yes, yes, sir," Crevel replied; "but—" "But it surprises you, I suppose?" "I confess it." "It is true," Leon said, with a mocking smile, "that it is not my habit to drink." "That it is not," said Crevel. "Well, I am going to take to it, that's all. And what do you find surprising in that?"
- 75. "Nothing, of course." "Then bring me what I asked for." "Directly, directly, captain." "On my soul, something extraordinary is taking place," Crevel said to himself as he descended to his bar. "The captain never had a very agreeable way with him, but, on the word of Crevel, I never saw him as he is tonight; it would be dangerous to touch him with a pair of tongs. What can have happened to him? Ah, stuff, it concerns him, after all: and then, who knows; perhaps he is on the point of becoming a drunkard." After this aside, the worthy landlord manufactured a splendid bowl of punch, which he carried up to Leon so soon as it was ready. "There," he said, as he placed the bowl on the table; "I think that will please you, captain." "Thanks! but what is this?" Leon said, as he looked at what Crevel had brought—"there is only one glass." "Why, you are alone." "That is true; but I trust you will do me the pleasure of drinking with me." "I should be most unwilling, captain, to deprive myself of the honour of drinking with you, but—" Crevel, through his stupefaction, was unable to complete his sentence, for the invitation which the captain gave him surprised him beyond all expression. Let us add that it was the first time such an honour had been done him. "In that case bring a glass for yourself." Crevel, without further hesitation, fetched the glass, and seated himself facing the captain. "Now, my dear Crevel," Leon said, as he dipped into the bowl and filled the glasses to the brim, "here's to your health, and let us talk."
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