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Nanoproteomics Methods and Protocols 1st Edition Jaco
C. Knol Digital Instant Download
Author(s): Jaco C. Knol, Connie R. Jimenez (auth.), Steven A. Toms, Robert J.
Weil (eds.)
ISBN(s): 9781617793196, 1617793191
Edition: 1
File Details: PDF, 7.05 MB
Year: 2011
Language: english

ME T H O D S I N MO L E C U L A R BI O L O G Y ™
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Nanoproteomics
Methods and Protocols
Edited by
Steven A.Toms
Department of Neurosurgery, Geisinger Health Systems, Danville, PA, USA
Robert J.Weil
Rose Ella Burkhardt Brain Tumor and Neuro-Oncology Center, Department of Neurosurgery,
The Neurological Institute, Cleveland Clinic, Cleveland, OH, USA

Editors
Steven A. Toms, MD, MPH, FACS
Department of Neurosurgery
Geisinger Health Systems
Danville, PA, USA
satoms@geisinger.edu
Robert J. Weil, MD
Rose Ella Burkhardt Brain Tumor
and Neuro-Oncology Center
Department of Neurosurgery
The Neurological Institute
Cleveland Clinic
Cleveland, OH, USA
weilr@ccf.org
ISSN 1064-3745 e-ISSN 1940-6029
ISBN 978-1-61779-318-9 e-ISBN 978-1-61779-319-6
DOI 10.1007
/978-1-61779-319-6
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2011936012
© Springer Science+Business Media, LLC 2011
All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the
publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA),
except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or
hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.
Printed on acid-free paper
Humana Press is part of Springer Science+Business Media (www.springer.com)

v
Preface
As the rise and evolution of two-dimensional gel electrophoresis began to allow the large-scale
analysis of proteins in the 1990s, the term “proteome” was more widely used to characterize
the entire set of proteins in an organism, and “proteomics,” the analysis of these proteins.
Nanotechnology, the study of manipulating matter less than 1/10 of a micron in diameter,
had its origins a decade or two decades earlier as scientists began to manipulate and engineer
substances on a molecule-by-molecule basis. However, it has been only within the past
decade that proteomics and nanotechnology have become mature, distinct endeavors and
have begun a scientific courtship; the nascent merger of the two disciplines now permits
precision in the separation, identification, and quantification of rare protein species.
As two relatively new fields of study, proteomics and nanotechnology have developed
in parallel to allow increased precision in the identification of posttranslational protein
modifications as well as to allow more automated isolation and detection of uncommon
proteins in serum and tissues. Despite the rapid advances of the past decade, proteomics has
not yet approached the scale and automation of its sister discipline, genomics, where the
ability to identify and quantitate rare transcripts has allowed rapid advances in the under-
standing of gene expression patterns in human disease.
Advances in nanotechnology, however, have begun to allow researchers to identify
low-abundance proteins in samples using techniques that rely upon nanoparticles, nano-
structured devices, and nanoscale separation techniques. When advances in nanotechnol-
ogy have been coupled with those made in protein identification and isolation, these
nanoproteomic technologies have enabled more effective multiplexing and improvements in
signal-to-noise ratio when compared to the techniques they have replaced. Taken together,
these methods have significantly enhanced the detection of low-abundance proteins and
posttranslational modifications in cellular lysates and tissue samples.
In this rapidly evolving field, Nanoproteomics: Methods and Protocols attempts to orga-
nize and collect technical advances from leaders in the field to make them more readily
available and understandable to those who are attempting to incorporate nanotechnologic
techniques into their proteomic research. This volume does not attempt to be encyclopedic
but rather seeks to capture a snapshot of those techniques in nanoscale separation and
nanotechnology-enhanced detection of rare proteins that will provide the foundation for
any group interested in developing or enhancing their understanding and methodological
expertise in the burgeoning field of nanoproteomics. This should serve both to aid in stan-
dardization of protocols as well as to inspire readers as to how these methods might advance
their own efforts in the study of the proteome.
This book is organized into five parts. In the first part, “Preliminary Sample Preparation,”
we highlight novel protocols for preparing biological samples necessary for formal proteomic
analysis. Part two, the largest of the five, describes a variety of nanoscale fluidic devices and
methods to aid in the enrichment of signal in separation and the analysis of proteomic
samples. Parts three and four cover nanostructured surfaces and nanomaterials that are used

vi Preface
to enrich protein signals and identify low signal proteins in the background of more
abundant proteins, which are present in most raw samples analyzed in proteomics analysis.
The final part deals with the aim of many developing nanoproteomic techniques to detect
and understand protein and proteomic alterations specific to human pathology.
The protocols useful in proteomics and nanotechnology have varied widely and are
often difficult to incorporate or expensive to try. It is the hope that these procedures, culled
from the laboratories of leaders in the field of nanoproteomics, will help in their standard-
ization and proliferation, leading to more wide-scale adoption. As these techniques and the
data derived from their use multiply, nanoproteomic applications should lead to a more
thorough comprehension of protein derangements in disease states, identifying potential
interventions for therapies in diseases as diverse as cancer, cardiovascular diseases, and
metabolic disorders.
Danville, PA Steven A. Toms
Cleveland, OH Robert J. Weil

vii
Acknowledgements
The editors acknowledge the book cover contribution by authors, Dr. Chia-Wen Tsao and
Dr. Don L. DeVoe.
Also, Drs. Weil and Toms would like to acknowledge the invaluable contribution by
Marian Repella Kozak in shepherding the editorial process as well as our wives, Stacie and
Alexandra in their support.

ix
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
PART I PRELIMINARY SAMPLE PREPARATION
1 MALDI-TOF Serum Profiling Using Semiautomated Serum Peptide
Capture with Magnetic Reversed Phase (C18) Beads. . . . . . . . . . . . . . . . . . . . . . . . 3
Jaco C. Knol and Connie R. Jimenez
2 Proteomics of Epithelial Lining Fluid Obtained by Bronchoscopic
Microprobe Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Lorenza Franciosi, Natalia Govorukhina, Nick ten Hacken,
Dirkje Postma, and Rainer Bischoff
PART II NANOSCALE SEPARATION
3 Protein Identification Using Nano-HPLC-MS: ESI-MS
and MALDI-MS Interfaces. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Rui Vitorino, Jana Krenkova, Frantisek Foret, Pedro Domingues,
and Francisco Amado
4 Three-Dimensional Peptide Fractionation for Highly Sensitive Nanoscale
LC-Based Shotgun Proteomic Analysis of Complex Protein Mixtures . . . . . . . . . . . 47
Sricharan Bandhakavi, Todd W. Markowski, Hongwei Xie,
and Timothy J. Griffin
5 Nanospray Ion Mobility Mass Spectrometry of Selected High Mass Species. . . . . . . 57
Iain Campuzano and Kevin Giles
6 Nanoelectrospray-MSn
of Native and Permethylated Glycans. . . . . . . . . . . . . . . . . . 71
Christina Bleckmann, Hildegard Geyer, and Rudolf Geyer
7 N-Linked Global Glycan Profiling by NanoLC Mass Spectrometry . . . . . . . . . . . . . 87
Michael S. Bereman and David C. Muddiman
8 Applications of Nanoscale Liquid Chromatography Coupled to Tandem Mass
Spectrometry in Quantitative Studies of Protein Expression,
Protein–Protein Interaction, and Protein Phosphorylation . . . . . . . . . . . . . . . . . . . 99
Metodi V. Metodiev
9 Nano LC–MS/MS: A Robust Setup for Proteomic Analysis . . . . . . . . . . . . . . . . . . 115
Marco Gaspari and Giovanni Cuda
10 Nanofluidic Devices for Rapid Continuous-Flow Bioseparation. . . . . . . . . . . . . . . . 127
Pan Mao and Jianping Fu
11 Quantifying Attomole Amounts of Proteins from Complex Samples
by Nano-LC and Selected Reaction Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Thomas Fröhlich and Georg J. Arnold

x Contents
PART III NANOMATERIALS: NANOSTRUCTURED SURFACES
12 A Sample Preparation Method for Gold Nanoparticle-Assisted
Laser Desorption/Ionization Time-of-Flight Mass Spectrometry . . . . . . . . . . . . . . 167
Yen-Hsiu Lin and Wei-Lung Tseng
13 Nanostructured TiO2
Thin Films for Phosphoproteomics Studies
with MALDI Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Federico Torta, Matteo Fusi, Carlo S. Casari, Andrea Li Bassi,
and Angela Bachi
14 Nanofilament Silicon for Matrix-Free Laser Desorption/Ionization
Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Chia-Wen Tsao and Don L. DeVoe
15 Protein Nanoarrays for High-Resolution Patterning of Bacteria
on Gold Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Cait Costello, Jan-Ulrich Kreft, Christopher M. Thomas,
and Paula M. Mendes
PART IV NANOMATERIALS: NANOSCALE TOOLS FOR PROTEOMIC DISCOVERY
16 Engineered Multifunctional Nanotools for Biological Applications . . . . . . . . . . . . . 203
Mohammed Ibrahim Shukoor, Muhammad Nawaz Tahir, Thomas Schladt,
Wolfgang Tremel, Zhiqun Zhang, Kevin K. Wang, and Firas H. Kobeissy
17 Selective Capture of Phosphopeptides by Zirconium
Phosphonate-Magnetic Nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Liang Zhao, Ren’an Wu, and Hanfa Zou
18 Nanowire Biosensors for Label-Free, Real-Time, Ultrasensitive
Protein Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Gengfeng Zheng and Charles M. Lieber
19 Generation of Anti-infectome/Anti-proteome Nanobodies. . . . . . . . . . . . . . . . . . . 239
Gholamreza Hassanzadeh-Ghassabeh, Dirk Saerens, and Serge Muyldermans
PART V NANOTECHNOLOGY: TECHNIQUES IN PROTEOMICS – POTENTIAL
APPLICATION FOR HEALTH AND DISEASE
20 Isolation, Propagation, and Analysis of Biological Nanoparticles . . . . . . . . . . . . . . . 263
Michael P. Linnes, Farooq A. Shiekh, Larry W. Hunter,
Virginia M. Miller, and John C. Lieske
21 Functionalized Soluble Nanopolymers for Phosphoproteome Analysis. . . . . . . . . . . 277
Anton Iliuk, Keerthi Jayasundera, Rachel Schluttenhofer, and W. Andy Tao
22 Elucidating Structural Dynamics of Integral Membrane Proteins on Native
Cell Surface by Hydroxyl Radical Footprinting and Nano LC-MS/MS . . . . . . . . . . 287
Yi Zhu, Tiannan Guo, and Siu Kwan Sze
23 Application of Electrostatic Repulsion Hydrophilic Interaction Chromatography
to the Characterization of Proteome, Glycoproteome, and Phosphoproteome
Using Nano LC–MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
Piliang Hao, Huoming Zhang, and Siu Kwan Sze
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

xi
Contributors
FRANCISCO AMADO s Department of Chemistry, Mass Spectrometry Center, QOPNA,
University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal
GEORG J. ARNOLD s Laboratory for Functional Genome Analysis LAFUGA,
Gene Center, Ludwig-Maximilians-University, Munich, Germany
ANGELA BACHI s Division of Genetics & Cell Biology, San Raffaele Scientiﬁc Institute,
Milan, Italy
SRICHARAN BANDHAKAVI s Department of Biochemistry, Molecular Biology
and Biophysics, University of Minnesota, Minneapolis, MN, USA
ANDREA LI BASSI s Dipartimento di Energia and NEMAS, Center for NanoEngineered
Materials and Surfaces, Politecnico di Milano, Milan, Italy; Center for Nano Science
and Technology of IIT at PoliMI, Milan, Italy
MICHAEL S. BEREMAN s Department of Genome Sciences, University of Washington,
Seattle, WA, USA
RAINER BISCHOFF s Department of Pharmacy, Analytical Biochemistry,
University of Groningen, Groningen, The Netherlands
CHRISTINA BLECKMANN s Institute of Biochemistry, Faculty of Medicine,
University of Giessen, Giessen, Germany
IAIN CAMPUZANO s Waters Corporation, Manchester, UK
CARLO S. CASARI s Dipartimento di Energia and NEMAS, Center for NanoEngineered
Materials and Surfaces, Politecnico di Milano, Milan, Italy;
Center for Nano Science and Technology of IIT@PoliMI, Milan, Italy
CAIT COSTELLO s School of Chemical Engineering, University of Birmingham,
Edgbaston, Birmingham, UK
GIOVANNI CUDA s Laboratory of Proteomics and Mass Spectrometry,
Department of Experimental and Clinical Medicine, “Magna Græcia”
University of Catanzaro, Catanzaro, Italy
DON L. DEVOE s Department of Mechanical Engineering, University of Maryland,
College Park, MD, USA
PEDRO DOMINGUES s Department of Chemistry, Mass Spectrometry Center, QOPNA,
FRANTISEK FORET s Institute of Analytical Chemistry of the ASCR, Brno, Czech Republic
LORENZA FRANCIOSI s Department of Pharmacy, Analytical Biochemistry,
THOMAS FRÖHLICH s Laboratory for Functional Genome Analysis LAFUGA,
Gene Center, Ludwig-Maximilians-University, Munich, Germany
JIANPING FU s Department of Mechanical Engineering and Biomedical Engineering,
University of Michigan, Ann Arbor, MI, USA
MATTEO FUSI s Dipartimento di Energia and NEMAS, Center for NanoEngineered
Materials and Surfaces, Politecnico di Milano, Milan, Italy

xii Contributors
MARCO GASPARI s Laboratory of Proteomics and Mass Spectrometry,
Department of Experimental and Clinical Medicine, “Magna Græcia”
University of Catanzaro, Catanzaro, Italy
HILDEGARD GEYER s Institute of Biochemistry, Faculty of Medicine,
University of Giessen, Giessen, Germany
RUDOLF GEYER s Institute of Biochemistry, Faculty of Medicine, University of Giessen,
Giessen, Germany
KEVIN GILES s Waters Corporation, Wythenshawe, Manchester, UK
NATALIA GOVORUKHINA s Department of Pharmacy, Analytical Biochemistry,
TIMOTHY J. GRIFFIN s Department of Biochemistry, Molecular Biology and Biophysics,
University of Minnesota, Minneapolis, MN, USA
TIANNAN GUO s School of Biological Sciences, Nanyang Technological University,
Singapore, Singapore
PILIANG HAO s School of Biological Sciences, Nanyang Technological University,
Singapore, Singapore
GHOLAMREZA HASSANZADEH-GHASSABEH s Department of Molecular and Cellular
Interactions, VIB, Brussels, Belgium; Laboratory of Cellular and Molecular
Immunology, Vrije Universiteit Brussel, Brussels, Belgium
LARRY W. HUNTER s Department of Surgery, Mayo Clinic, Rochester, MN, USA
ANTON ILIUK s Departments of Biochemistry, Chemistry, and Medicinal Chemistry,
and Molecular Pharmacology, Purdue Center for Cancer Research,
Purdue University, West Lafayette, IN, USA
KEERTHI JAYASUNDERA s Departments of Biochemistry, Chemistry, and Medicinal
Chemistry, and Molecular Pharmacology, Purdue Center for Cancer Research,
CONNIE R. JIMENEZ s OncoProteomics Laboratory, Department of Medical Oncology,
VU University Medical Center, Amsterdam, The Netherlands
JACO C. KNOL s OncoProteomics Laboratory, Department of Medical Oncology,
VU University Medical Center, Amsterdam, The Netherlands
FIRAS H. KOBEISSY s Center of Innovative Research, Banyan Biomarkers, Inc.,
Alachua, FL, USA; Center for Neuroproteomics and Biomarkers Research,
Department of Psychiatry, McKnight Brain Institute, University of Florida,
Gainesville, FL, USA
JAN-ULRICH KREFT s School of Biosciences, University of Birmingham, Edgbaston,
Birmingham, UK
JANA KRENKOVA s Institute of Analytical Chemistry of the ASCR, Brno, Czech Republic
CHARLES M. LIEBER s Department of Chemistry and Chemical Biology and Division of
Engineering and Applied Science, Harvard University, Cambridge, MA, USA
JOHN C. LIESKE s Division of Nephrology and Hypertension, College of Medicine,
Mayo Clinic, Rochester, MN, USA
YEN-HSIU LIN s Department of Chemistry, National Sun Yat-sen University,
Kaohsiung, Taiwan
MICHAEL P. LINNES s Division of Nephrology and Hypertension,
Department of Medicine, Mayo Clinic, Rochester, MN, USA

xiii
Contributors
PAN MAO s Lawrence Berkeley National Laboratory, Berkeley, CA, USA
TODD W. MARKOWSKI s Center for Mass Spectrometry and Proteomics,
University of Minnesota, Minneapolis, MN, USA
PAULA M. MENDES s School of Chemical Engineering, University of Birmingham,
METODI V. METODIEV s Department of Biological Sciences, University of Essex,
Colchester, UK
VIRGINIA M. MILLER s Department of Physiology and Surgery, College of Medicine,
Mayo Clinic, Rochester, MN, USA
DAVID C. MUDDIMAN s Department of Chemistry, North Carolina State University,
Raleigh, NC, USA
SERGE MUYLDERMANS s Department of Molecular and Cellular Interactions,
VIB, Brussels, Belgium; Laboratory of Cellular and Molecular Immunology,
Vrije Universiteit Brussel, Brussels, Belgium
DIRKJE POSTMA s Department of Pulmonary Diseases, University Medical Center
Groningen, University of Groningen, Groningen, The Netherlands
DIRK SAERENS s Department of Molecular and Cellular Interactions, VIB,
Brussels, Belgium; Laboratory of Cellular and Molecular Immunology,
Vrije Universiteit Brussel, Brussels, Belgium
THOMAS SCHLADT s Institut für Anorganische Chemie und Analytische Chemie,
Johannes Gutenberg-Universität Mainz, Mainz, Germany
RACHEL SCHLUTTENHOFER s Departments of Biochemistry, Chemistry,
and Medicinal Chemistry, and Molecular Pharmacology, Purdue Center
for Cancer Research, Purdue University, West Lafayette, IN, USA
FAROOQ A. SHIEKH s Division of Nephrology and Hypertension, Mayo Clinic,
Rochester, MN, USA
MOHAMMED IBRAHIM SHUKOOR s Institut für Anorganische Chemie und Analytische
Chemie, Johannes Gutenberg-Universität Mainz, Mainz, Germany
SIU KWAN SZE s School of Biological Sciences, Nanyang Technological University,
Singapore
MUHAMMAD NAWAZ TAHIR s Institut für Anorganische Chemie und Analytische
Chemie, Johannes Gutenberg-Universität Mainz, Mainz, Germany
W. ANDY TAO s Departments of Biochemistry, Chemistry, and Medicinal Chemistry,
and Molecular Pharmacology, Purdue Center for Cancer Research,
NICK TEN HACKEN s Department of Pulmonary Diseases, University Medical Center
Groningen, University of Groningen, Groningen, The Netherlands
CHRISTOPHER M. THOMAS s School of Biosciences, University of Birmingham,
FEDERICO TORTA s Division of Genetics & Cell Biology, San Raffaele Scientiﬁc
Institute, Milan, Italy; Mechanobiology Institute and Lipid Proﬁles,
Centre for Life Sciences, National University of Singapore, Singapore
WOLFGANG TREMEL s Institut für Anorganische Chemie und Analytische Chemie,
Johannes Gutenberg-Universität Mainz, Mainz, Germany
CHIA-WEN TSAO s Department of Mechanical Engineering, National Central
University, Jhongli, Taiwan

xiv Contributors
WEI-LUNG TSENG s Department of Chemistry, National Sun Yat-sen University,
Kaohsiung, Taiwan
RUI VITORINO s Department of Chemistry, Mass Spectrometry Center, QOPNA,
KEVIN K. WANG s Center of Innovative Research, Banyan Biomarkers, Inc., Alachua,
FL, USA; Center for Neuroproteomics and Biomarkers Research,
Department of Psychiatry, McKnight Brain Institute, University of Florida,
Gainesville, FL, USA
REN’AN WU s National Chromatographic R & A Center, CAS Key Laboratory
of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics,
Chinese Academy of Sciences, Dalian, China
HONGWEI XIE s Waters Corporation, Milford, MA, USA
HUOMING ZHANG s School of Biological Sciences, Nanyang Technological University,
Singapore
ZHIQUN ZHANG s Center of Innovative Research, Banyan Biomarkers, Inc, Alachua,
FL, USA
LIANG ZHAO s National Chromatographic R & A Center, CAS Key Laboratory
GENGFENG ZHENG s Laboratory of Advanced Materials, Department of Chemistry,
Fudan University, Shanghai, China
YI ZHU s School of Biological Sciences, Nanyang Technological University, Singapore,
Singapore
HANFA ZOU s National Chromatographic R & A Center, CAS Key Laboratory

Part I
Preliminary Sample Preparation

3
Steven A. Toms and Robert J. Weil (eds.), Nanoproteomics: Methods and Protocols, Methods in Molecular Biology, vol. 790,
DOI 10.1007/978-1-61779-319-6_1, © Springer Science+Business Media, LLC 2011
Chapter 1
MALDI-TOF Serum Profiling Using Semiautomated Serum
Peptide Capture with Magnetic Reversed Phase (C18) Beads
Jaco C. Knol and Connie R. Jimenez
Abstract
Mass spectrometry can be used to generate diagnostic peptide peak profiles “signatures” of serum samples.
Peak profiles can be used to compare different sera and correlate samples (i.e., patient groups) with clinical
data to assist in diagnosis, monitoring, and/or prediction.
We describe the semiautomated capture of serum peptides/small proteins with magnetic beads that
harbor C18 alkyl chains, the deposition of captured material on a target plate for MALDI-TOF mass spec-
trometry, as well as, general guidelines for data acquisition. We also include, in a separate note, a short
manual version of the capture procedure. Overall, the serum sample processing protocol, reported here, is
reproducible and robust. In conjunction with MALDI-TOF mass spectrometry, this protocol allows for
the profiling of several hundreds of serum peptides.
Key words: Magnetic beads, C18, Reproducibility, Automation, MALDI-TOF MS, Serum, Peptide
profiling
Human body fluids, that can be collected noninvasively, have been
an attractive source for biomarker discovery approaches. Blood
samples are especially easy to obtain, and their contents are likely
to reflect the physiological and pathophysiological state of the
whole human body. Blood reaches all the organs and tissues in
the body, through secretion or shedding, and enriches the total
spectrum of proteins present in the bloodstream. Indeed, in recent
studies, we and other researchers have successfully combined serum
peptide profiling by mass spectrometry (MS) with bioinformatics
and have established distinctive serum polypeptide MS patterns
that correlate with cancer types and clinically relevant outcomes
1. Introduction

4 J.C. Knol and C.R. Jimenez
(1–3). Moreover, researchers have provided a direct link between
the peptide marker profiles of cancer and differential protease activity,
which suggests that the patterns may have clinical utility as surro-
gate markers for detection and classification of cancer.
In the present procedure, peptides and small proteins that are
present in serum are captured by microparticle beads that harbor
C18 alkyl chains. Binding occurs through hydrophobic “reversed
phase” interactions between the C18 chains on the beads (hydro-
phobic “stationary phase”) and hydrophobic patches on peptides/
small proteins in the serum (hydrophilic “mobile phase”). By using
magnetic beads, the beads and their cargo can be collected with
magnets and serially transferred to washing and elution solutions
that allow automation with a dedicated robot. In this way, peptides/
small proteins are purified that are separate from substances, such
as salts, that interfere with the analysis of mass spectrometers, which
are equipped with a matrix-assisted laser desorption ionization
(MALDI) source that allows informative MALDI mass spectra to
be generated. An outline of the general workflow (see Fig. 1) has
been described in several papers (1, 4–7).
Before bead capturing, sera are first pretreated by adding non-
polymeric detergent and organic acid to improve peptide solubili-
zation that, consequently, will increase the number of detected MS
peaks. Then, for automated peptide capture, a series of 96-well
plates is manually filled with magnetic C18 bead suspension, pretreated
serum samples, washing solution, and elution solution, respec-
tively. Subsequently, the plates are placed in a KingFisher 96®
robot
that is equipped with a turntable of eight docking positions
for 96-well plates, as well as, a vertically movable head with 96
magnetic rods. The latter, enveloped by a plastic cover (comb),
Fig. 1. Body fluid profiling workflow. Body fluid samples (e.g., serum or cerebrospinal fluid, CSF) are mixed with magnetic
(C18) beads, and peptides (and/or proteins) are allowed to adsorb. Contaminants can then be washed away, and peptides
eluted.Aliquots containing eluted peptides are analyzed by MALDI-TOF mass spectrometry, and the data are then subjected
to some form of bioinformatics analysis (e.g., principal component analysis or cluster analysis) (10). The biofluid peptide
capture procedure is semiautomated as described in this chapter.

5
1 Semi-Automated MALDI-TOF Serum Profiling
can be inserted into the well of any plate positioned beneath the
head. The beads are automatically collected, equilibrated, incu-
bated with samples, washed, and eluted into 96 different well plates
through the rotation of the appropriate plate under the movable
head. Then, the beads are transferred from one plate to another by
the comb-covered magnetic rods, which are also used as a mixing
device. Eluates are manually transferred to microcentrifuge tubes,
and a small aliquot of each eluate is combined with a MALDI
matrix solution and spotted on a stainless steel target plate for
automated analysis on a MALDI-TOF mass spectrometer.
The C18-Dynabead-based serum sample processing protocol,
reported here, is reproducible (6). Examples of serum peptide profiles
of different aliquots in the same batch of control sera are shown
(see Fig. 2a–c), which are highly similar when measured within the
same experiment and even between experiments with a 2 year
timeframe between experiments that underscores the robustness of
Fig. 2. (a–c) Reproducibility of serum peptide profiles. MALDI-TOF MS spectra that are obtained for aliquots of the same
in-house collected serum sample are shown. Two independent aliquots are processed as described and measured in
experiments 1 and 2. Two years later, aliquots are processed and measured in experiment 2. Overall peak patterns are
highly similar within experiments and in between experiments. For details on %CV values, the reader is referred to Jimenez
et al.(6). Different zoom views are shown (a) contains no zoom, (b) is 10× zoom, and (c) is 100× zoom to appreciate the
reproducibility of the peak patterns in the overall profile (a), which is dominated by a few abundant species, as well as, the
patterns in the intermediate (b) and low signal intensity (c) ranges.
Mass (m/z)
%
Intensity
100
100
100
800 1440 2080 2720 3360 4000
800 1440 2080 2720 3360 4000
800 1440 2080 2720 3360 4000
800 1440 2080 2720 3360 4000
100
a
CON Sample
2009
CON Sample
2007
CON Sample
2007
CON Sample
2009

Fig. 2. (continued)

7
the procedure. In conjunction with the KingFisher 96®
, the whole
serum peptide capture procedure is high throughput, and takes
only ~20 min per batch isolation of 96 samples, thereby, facilitating
large-scale disease profiling studies.
Solutions can be used for 2 months unless stated otherwise.
Trifluoroacetic acid (TFA) and acetonitrile (ACN) are volatile;
therefore, prepared in fume hood.
1. Clinical serum samples: collect serum in a standardized fashion
with 1 h clotting time, aliquot in 50–300 PL portions, and
immediately store at −80°C, and subject to a maximum of two
freeze–thaw cycles. It is essential to adhere to a fixed sample
collection and handling protocol (see Note 1). In the present
setup, a minimum of 20 PL serum is needed.
2. Standard serum control: collect and aliquot a large batch of
known control (CON) serum from healthy volunteer(s) and
store at −80°C (see Note 2). For each experiment, the CON
serum sample is included in four wells that are distributed over
a KingFisher 96®
plate containing clinical serum samples in all
other wells (see Fig. 3). MALDI spectra that are generated
from these CON aliquots will allow quality control and may
serve as a reference when linking different experiments.
3. C18 beads: monodisperse magnetic beads derivatized with
octadecyl (C18) chains, 1-Pm Dynabeads®
RPC18 (Invitrogen,
Breda, The Netherlands).
4. MALDI matrix: 6.2 mg/mL D-cyano-4-hydroxycinnamic acid
in36%methanol,56%ACN(AgilentTechnologies,Amstelveen,
The Netherlands).
5. NaCl/TFA: 200 mM NaCl and 0.1% (v/v) TFA in double-
distilled water.
6. OG/TFA (make fresh each time): 0.15% (w/v) n-octyl-E-d-
glucoside and 0.5% (v/v) TFA in double-distilled water.
7. 0.1% TFA: 0.1% (v/v) TFA in double-distilled water.
8. 50% ACN: 50% (v/v) ACN in double-distilled water.
9. KingFisher 96®
Magnetic Particle Processor: robot for auto-
mated magnetic bead-based processing of liquid samples in
96-well plates (Thermo Fisher Scientific, Breda, The
Netherlands).
10. KingFisher 96®
KF Microplates: 96-well microplates (200 PL)
for use with 96 KF head (Thermo Fisher Scientific).
2. Materials

11. KingFisher 96®
Tip Comb: tip comb for use with a 96 KF head
(Thermo Fisher Scientific).
12. Solution Tray: transfer tray for solutions with a multichannel
pipette that should not give off polymers (see Note 3).
13. Microcentrifuge tubes: low-adsorbing and low polymer-releasing
(see Note 3) Safe-Lock tubes (Eppendorf, distributor VWR,
Amsterdam, The Netherlands).
14. Multiwell Plate Shaker: vortex/shaker to mix solutions in
96-well plates, such as Vortex-Genie2 (Scientific Industries,
distributor VWR, Amsterdam, The Netherlands).
15. MALDI Target Plate: appropriate stainless steel target plate
for MALDI-MS analysis; we use Opti-TOF 384 Well Inserts
for the 4800 MALDI-TOF/TOF Analyzer (ABSCIEX,
Nieuwerkerk a/d IJssel, The Netherlands).
1. Create an Excel document such as “Serum Profiling Sample
List dd-mm-yyyy,” linking numbers (1, 2, 3, etc.) to the preex-
istent sample IDs/codes.
3. Methods
3.1. Preparation
(See Note 4)
96
95
94
93
92
CON
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90
89
88
87
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H
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F
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8
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3
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A
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10
9
8
7
6
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4
3
2
1
Fig. 3. KingFisher 96®
plate well lay-out. Four aliquots of a (CON) serum are included in the procedure for quality control
and interexperiment comparison purposes. They are distributed over the plate to monitor possible position-dependent
differences in performance of the procedure. The clinical serum samples are randomized over all other positions in the
plate so as to minimize any biases. Eluates are numbered by the corresponding well number as indicated here.

9
2. Sample randomization is essential in serum profiling (see Note 5).
Make a randomized allocation scheme for each 96-well plate to
be analyzed in following steps 3–5.
3. Make a 96-well plate scheme, and allocate four aliquots of the
control serum sample (CON) to four wells distributed over the
plate (see Fig. 3).
4. Randomize 92 clinical serum samples over the remaining
well positions. This can be done with any program such as
the Research Randomizer (http://www.randomizer.org).
Generate a random distribution of the numbers 1–92 (e.g., 68,
84, 32, 41, etc.). Using this series, look up the corresponding
sample IDs/codes in the “Serum Profiling Sample List dd-mm-
yyyy” document and enter into the plate scheme, going left–
right and up–down, from well 1 (well A1) to well 96 (well
H12), while skipping the four wells that have already been
assigned to the control serum sample aliquots.
5. In the “Serum Profiling Sample List dd-mm-yyyy” file, enter the
plate well number to which each sample has been allocated.
6. Label 0.5-mL Eppendorf microcentrifuge tubes with a num-
bered sticker of 1–96 on the lid. These will be used to store
peptide eluates, and the numbers correspond to plate well
numbers as indicated above, not sample numbers. Place the
numbered tubes in a tube holder, in a similar 8×12 array as
indicated (see Fig. 3). The row spacing in the holder should
match the tip spacing of a multichannel pipette.
7. Label 8 KingFisher 96®
plates with a number at the side as
plates 1–8 as shown (see Table 1), and place a KingFisher 96®
tip comb in plate 8.
1. Retrieve and thaw clinical serum samples and CON serum on
ice as early as possible (see Note 8). If not done before (e.g., in
the case of analyzing third-party samples), then, note that the
samples are clearly more red than just slightly “orange.” These
are hemolytic sera and are not useful for signature development
(see Note 9).
2. Thoroughly resuspend magnetic C18 beads in the stock vial by
shaking on a vortex for t2 min.
3. Using a multichannel pipette and a solution tray, pipette as
follows: (1) 200 PL 0.1% TFA in all wells of plates 4, 5, and 6;
(2) 200 PL NaCl/TFA in all wells of plate 2; and (3) 120 PL
NaCl/TFA in all wells of plate 1.
4. Add 80 PL C18 bead suspension (1 mg beads) to all wells of
plate 1.
5. On ice, using the prepared (see Subheading 3.1, step 2) random-
ized plate filling scheme, pipette 20 PL of the appropriate
serum sample on the bottom of the wells of plate 3.
3.2. Semiautomated
Serum Peptide
Capture with Magnetic
C18 Beads in a
KingFisher 96®
Robot
(See Notes 6 and 7)

6. Remove plate 3 from the ice, dry bottom with tissue, and leave
at room temperature for 5 min.
7. Use a multichannel pipette and a solution tray, add 40 PL OG/
TFA to all wells of plate 3. Mix gently by slowly pipetting up
and down to prevent excessive foaming (see Note 10).
8. Gently shake plate 3 on a 96-well plate shaker for 5 min at
room temperature so that the contents of the wells are not
spilled; position 4.5–5 on a Vortex-Genie 2.
9. Switch on KingFisher 96®
robot and select the appropriate
protocol with the and buttons (a working KingFisher pro-
tocol can be obtained from the authors); load the protocol
from a computer linked to the KingFisher 96®
through an
RS-232C cable.
10. Load plates 1–6 and plate 8 with a tip comb in their proper
docking positions on the KingFisher 96®
turntable (i.e., well
A1 facing inward-left). Use < and > buttons to rotate the turntable
left–right in order to bring docking positions 1–8 to the front
in the loading position.
11. Just before starting the KingFisher run (see Note 11), using a
multichannel pipette and a solution tray, add 40 PL 50% ACN
to all wells of plate 7, load this plate in its proper docking position
on the KingFisher turntable, and shut the sliding door.
Table 1
Summary overview of the different 96-well plates used in the KingFisher
96®
-mediated serum peptide capture procedure
Plate To be loaded with Purpose
1 Magnetic beads in NaCl/TFA Collect and prewash beads
2 200 PL NaCl/TFA (Thermo Fisher Scientiﬁc,
Breda, The Netherlands)
Equilibrate beads
3 20 PL serum and 40 PL OG/TFA (Thermo Fisher
Scientiﬁc, Breda, The Netherlands)
Bind peptides/small proteins
to beads
4 200 PL 0.1% TFA (should not give off polymers,
see Note 1)
Wash beads and cargo
5 200 PL 0.1% TFA (see Note 1) (Eppendorf,
distributor VWR, Amsterdam, The Netherlands)
(should not give off polymers, see Note 1)
Wash beads and cargo
6 200 PL 0.1% TFA Wash beads and cargo
7 40 PL 50% ACN Elute peptides/small proteins
from beads
8 KingFisher 96®
Tip Comb Collect tip comb (cover)
of magnetic rods

11
12. Run the selected protocol on the KingFisher 96®
robot by
pressing START. Machine will ask for all plates; keep pressing
START.
13. Return all remaining serum to a −80°C freezer. After the cap-
ture procedure, they can be transiently stored at −20°C, but at
least at the end of the day, they should be returned to −80°C.
14. After completion of the KingFisher run, taking ~20 min, press
STOP, and immediately (see Note 11) transfer all eluates con-
tained in plate 7 to the numbered 0.5-mL Eppendorf tubes as
prepared (see Subheading 3.1, step 6). With the opened tubes
positioned in an 8×12 array similar to the wells of the 96-well
plate, transfer eluates with a multichannel pipette. Make sure
that all well numbers and tube numbers match, thus, maintain-
ing randomization.
15. Store the tubes with eluates at −20°C.
1. Place eluate tubes on ice.
2. Pipette 2 PL MALDI matrix solution into a series of 6–8
Eppendorf tubes.
3. Add 1 PL KingFisher (peptide) eluate to one of the tubes with
MALDI matrix; mix by gently pipetting up and down.
4. Then, spot 0.7 PL of the mix on a MALDI target plate. Make
duplicate spots for each eluate as exemplified for a 384-spot
target (see Fig. 4) (e.g., spotting series 1 for immediate MS
analysis and series 2 as a fail-safe backup). Maintain the order
of 1–96 of the eluates for the sake of sample randomization.
1. Tune the MALDI-MS instrument for optimal signal-to-noise
output. One important parameter is the laser intensity setting.
The serum needs higher intensity than calibrant peptides on
the 4800 MALDI-TOF/TOF platform.
2. Program a batch job that records MS spectra for all samples.
Generate spectra from spot series 1, leaving spot series 2
untouched as a backup in case things go awry with the mea-
surement of series 1, or for manual (MS/MS) measurements.
1. Assess whether all CON serum profiles are similar in terms of
peak intensity and peak counts (i.e., the number of different
peaks in the spectrum) and whether they are similar to previ-
ous experiments with the same CON serum (see Note 2).
2. Assess whether all clinical serum profiles are within the upper
detection limit of the apparatus (e.g., 100,000 counts on a
4800 MALDI-TOF/TOF). If there are mass spectra that
exhibit peaks exceeding the detection range, then measure the
second sample spot series with lower laser intensity so that all
peaks fall within the scalable detection range.
3.3. Eluate Spotting for
MALDI-TOF Profiling
(See Notes 11 and 12)
3.4. MALDI-MS
Profiling (See
Notes 13 and 14)
3.5. Data Analysis

3. Check all clinical serum profiles for clearly aberrant patterns.
Hemolytic serum samples give hemoglobin-derived peak
patterns, which do not contribute nor interfere with signature
development and therefore, are not useful for serum profiling
(see Note 9). Both salts that are present in serum, but removed
in the peptide capture procedure with hydrophobic C18 chains
and polymers with possible contaminants released by plastics,
can interfere with the MALDI-MS analysis due to a suppression
of peptide ion formation. In the case of polymers, replace the
peptide peak profile with a repetitive polymer-derived peak
pattern (see Note 3). Aberrant profiles should not be included
during signature development.
4. General steps in data preprocessing include peak extraction,
base line adjustment, spectrum alignment, etc. The reader is
referred to dedicated papers detailing the data mining (8, 9).
We employ the software package described by Pham and
Jimenez (9) that is designed to facilitate the complete data
analysis pipeline as follows: raw spectral data preprocessing,
study groups specification for comparison, statistical differential
analysis, peptide peaks visualization, and classification. The
software supports various external tools for these tasks.
72
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Spot
Series 1
Spot
Series 2
CON
CON
CON
CON
CON
CON
CON
CON
P
O
N
M
L
K
J
I
H
G
F
E
D
C
B
A
Fig. 4. MALDI target plate layout.A small aliquot of each of the eluates, resulting from the serum peptide capture procedure,
are spotted onto a 384-spot MALDI target plate, according to numbering in the KingFisher step. Thus, there is also a ran-
domization of samples on the MALDI plate. Eluates are spotted in duplicate (i.e., in Spot Series 1 and Spot Series 2) with
one series destined to be used for immediate MALDI-MS profiling in automated batch mode and the other series saved as
a backup in case of instrument problems with the first series or for manual MS/MS measurements on selected spots.

13
1. Adequate sample collection and handling is crucial to successful
serum profiling. Numerous “signatures,” which were described
in the past, could, in fact, be attributed to differences in clotting
time, room temperature handling time, storage temperature,
storage time, and freeze–thaw cycles. It is, therefore, essential
to stringently adhere to a standard collection protocol with
fixed clotting time (i.e., optimum: 1–2 h), immediate freezing
with a maximum of two freeze–thaw cycles, and sample handling
on ice during the protocol. Recently, a novel profiling strategy
was presented which virtually abolishes the above reproduc-
ibility problems (10) (see Note 15).
2. To assess proper functioning of the pipeline, it is important to
always take along the same CON serum sample. Processing
multiple aliquots of the same CON sample, which are distrib-
uted over the 96-well plate during serum peptide capture with
C18 beads and distributed over the MALDI target during MS
profiling, allows one to monitor intra-experiment reproduc-
ibility. Using the same control in all experiments, one can assess
the inter-experiment variability. The intra-experiment %CV
for normalized peak intensities typically average ~10%, and the
corresponding inter-experiment %CV is 31% on average (6). It
is important to monitor and control these variables.
3. Various brands and types of laboratory plastics can give off
polymers, especially when in contact with these organic solvents.
Polymers are detrimental for MS analysis, which suppress
peptide ion formation and replace the normal peptide peak
profile with a polymer-dominated, reiterating MS spectrum
that is useless for sample profiling purposes. We, therefore,
always use trusted polypropylene plastics as follows: microcen-
trifuge tubes, Eppendorf®
Safe-Lock™ tubes (VWR
International, Amsterdam, The Netherlands); 15 and 50-mL
tubes (Greiner Bio-One, Alphen a/d Rijn, The Netherlands).
4. Since it is important to perform serum peptide capture and
MALDI-MS profiling on the same day (see Note 14), processing
large sample collections is time consuming. Note that the com-
bination of sample retrieval, KingFisher-mediated peptide
capture, eluate spotting, and MALDI analysis may take one long
day. Therefore, it is advisable to finish preparations, if possible,
on the previous day.
5. Another source of bias, in addition to others that are mentioned
(see Note 1), may be introduced due to the different positions
of different samples in the KingFisher 96®
-well plate and on the
MALDI target plate. Therefore, sample randomization is cru-
4. Notes

cial. In the case of large sample collections measured over more
than one 96-well plate, we prefer longitudinal time series samples
of the same patient to be on the same plate.
6. Our serum peptide protocol is adapted for use with Dynabeads
RPC18 batches that contain 12.5 mg/mL beads by the addi-
tion of 1 mg beads per 20 PL serum sample. It can be easily
adapted to other brands of beads by adjusting the volumes
pipetted in plate 1 so that 1 mg beads are used (i.e., assuming
similar C18 loading and bead density). The procedure is
described for the full use of a 96-well plate, but the number of
used wells can be adjusted as required.
7. When testing the procedure for the first time, we suggest that
the serum peptide capture protocol be performed manually. In
short, the manual sample processing protocol (i.e., using same
volumes as described for automated capture) is as follows: (1)
serum and optional binding buffer (e.g., in our case, OG/TFA)
are mixed in microcentrifuge tubes, and C18 bead suspension
that is prewashed and equilibrated with NaCl/TFA is added,
mixed, and incubated for 2 min; (2) tubes are placed in a man-
ual magnetic particle separator such as a DynaMag (Invitrogen);
(3) the “supernatant” is aspirated; (4) tubes are removed from
the separator, and the wash solution of 0.1% TFA is added and
briefly mixed; (5) tubes are placed back into the separator with
wash solution, followed by aspiration of the “supernatant”; (6)
repeat washing procedure 3×; (7) after final washing step,
bound peptides and proteins are eluted by incubation with 50%
ACN for 2 min; (8) elution solution is collected by placing the
tubes in the separator, transferring the “supernatant” (i.e., pep-
tide eluate) to fresh tubes. This manual procedure may be used
in conjunction with a standard serum sample to optimize buf-
fer compositions and volumes in order to generate a protocol
that results in desirable peptide peak patterns and numbers (6).
8. Gentle thawing of large serum aliquots >100–200 PL on ice can
take 1 h or more. Therefore, thaw as early as possible, so that after
preparing the steps for the KingFisher plates (see Subheading 3.2,
steps 3 and 4), immediately proceed with the steps for loading
the serum samples (see Subheading 3.2, step 5).
9. Hemolysis, which results in the release of hemoglobin and
other contents from lysed erythrocytes into the serum, will
change the serum to a red color instead of a normal yellowish
color. With limited hemolysis, the serum will turn orange.
With more pronounced hemolysis, the color will turn dark red.
Samples with extensive hemolysis will not give rich, informative
MS profiles; instead, hemoglobin-derived peaks are prevalent
and are useless for signature development. One may decide to
exclude them from the experiment or use them for documen-
tation/verification or other purposes.

15
10. We have found that the nonionogenic detergent octyl glucoside
helps to solubilize certain serum peptides, so that the overall
peak count (i.e., number of peaks in a MALDI-MS spectrum)
is somewhat higher than in samples without the inclusion of
the detergent (6). One should take care not to cause excessive
foaming by vigorous pipetting or shaking. The TFA is included
to lower the pH so that protein precipitation is reduced (6).
11. ACN is a volatile compound. To ensure that the concentration
is right, which is important for peptide elution from the C18
beads, minimize the amount of time that the container is
opened. This also holds true for the ACN-containing matrix
solution. To minimize ACN evaporation from its wells, the
elution plate (plate 7) is prepared (see Subheading 3.2, step 1)
prior to starting the KingFisher run. It is also important to
collect the eluates from plate 7 and transfer them to the tubes
(see Subheading 3.1, step 6) as soon as the KingFisher run is
completed.
12. Eluate mixing, with MALDI matrix and spotting on the
MALDI target plate, can be done in sessions of about 6–8
samples at a time: pipetting matrix in 6–8 tubes and then, mix-
ing in and spotting eluate samples one at a time. Always keep
solvent evaporation to a minimum by quick pipetting and
immediate closure of tubes (see Note 11).
13. For MALDI-MS profiling, we use the 4800 MALDI-TOF/
TOF Analyzer (Ab Sciex LLC, Dublin, CA). Profiles generated
on different MALDI-MS platforms, with different lasers and
sensitivities, may differ significantly.
14. Perform the MALDI-MS measurement of series 1 on the same
day as the serum peptide capture (KingFisher run) to measure
as many low-intensity peaks in the MALDI-MS profile, as
possible, upon the storage of peptide eluates or unused MALDI
spots. These small peaks, which may contribute to discriminating
signatures, may no longer be detected if not performed on the
same day.
15. Recently, a novel strategy, Sequence-Specific Exopeptidase
Activity Test (SSEAT), has been presented by Villanueva et al.
(10, 11). Here, we do not look at the endogenously formed
peptide products (ENDO) but rather, at the action of the still-
active proteases on synthetic peptides that are added to the
serum. An isotopically labeled full-length peptide serves as a
degradable protease substrate (EXO) and is added together
with a set of nondegradable, doubly labeled synthetic peptides
that represent a ladder of degradation products (REF). The
latter serves as internal ratio standards for quantitation of the
various EXO degradation products (DEGR) that is generated
by the action of endogenous proteases on the single-labeled

EXO peptide after a specific incubation time at a specific
temperature, which yields multiple DEGR/REF ratios. The
ENDO, DEGR, and REF peptides of the same sequence are
separated in MS spectra that are due to the different degrees of
isotopic labeling. The SSEAT approach (i.e., spiking labeled
substrates for trace amounts of enzymatic activities) may allow
profiling in a background of high-abundant proteins, even
when using archived material, which has largely retained its
protease activities that are irrespective of the way the samples
are handled (see Note 1). The assay can be fully controlled and
tuned (i.e., peptide concentrations, incubation time, and tem-
perature) although it requires tuning for each new sample set.
It has been shown by Villanueva et al. (10) that, when using
labeled EXO/REF sets for three serum peptides (C3f, FPA-A,
and clusterin), multivariate analysis on all DEGR/REF ratios
can generate a significant signature for thyroid cancer with 94%
high sensitivity and 90% specificity.
References
1. Villanueva, J., Shaffer, D.R., Philip, J., et al.
(2006) Differential exoprotease activities confer
tumor-specific serum peptidome patterns. J Clin
Invest 116, 271–284.
2. Voortman, J., Pham, T.V., Knol, J.C., et al.
(2009) Prediction of outcome of non-small cell
lung cancer patients treated with chemotherapy
and bortezomib by time-course MALDI-
TOF-MS serum peptide profiling. Proteome Sci
7:34.
3. Karpova, M.A., Moshkovskii, S.A., Toropygin,
I.Y., et al. (2010) Cancer-specific MALDI-TOF
profiles of blood serum and plasma: biological
meaning and perspectives. J Proteomics 73,
537–551.
4. Villanueva, J., Philip, J., Entenberg, D., et al.
(2004) Serum peptide profiling by magnetic
particle assisted, automated sample processing
and MALDI-TOF mass spectrometry. Anal
Chem 76, 1560–1570.
5. De Noo, M.E., Tollenaar, R.A., Ozalp, A., et al.
(2005) Reliability of human serum protein pro-
files generated with C8 magnetic beads assisted
MALDI-TOF mass spectrometry. Anal Chem
77, 7232–7241.
6. Jimenez, C.R., El Filali, Z., Knol, J.C., et al.
(2007) Automated serum peptide profiling
using novel magnetic C18 beads off-line
coupledtoMALDI-TOF-MS(2007)Proteomics
Clin Appl 1, 598–604.
7. Villanueva, J., Lawlor, K., Toledo-Crow, R.,
et al (2006) Automated serum peptide profiling.
Nat Protoc 1, 880–891.
8. Villanueva, J., Philip J., DeNoyer, L., et al
(2007) Data analysis of assorted serum pepti-
dome profiles. Nat Protoc 2, 588–602.
9. Pham,T.V.,Jimenez,C.R.(2008)OplAnalyzer:
a toolbox for MALDI-TOF mass spectrometry
data analysis. In: Perner P., Salvetti O. (eds)
Advances in Mass Data Analysis of Images
and Signals in Medicine, Biotechnology,
Chemistry and Food Industry, MDA (2008),
Lect Notes Comput Sci 5108, 73–81. Springer,
Heidelberg.
10. Villanueva, J., Nazarian, A., Lawlor, K., et al.
(2008) A sequence-specific exopeptidase
activity test (SSEAT) for “functional” bio-
marker discovery. Mol Cell Proteomics 7,
509–518.
11. Villanueva, J., Nazarian, A., Lawlor, K.,
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1183.

17
Steven A. Toms and Robert J. Weil (eds.), Nanoproteomics: Methods and Protocols, Methods in Molecular Biology, vol. 790,
DOI 10.1007/978-1-61779-319-6_2, © Springer Science+Business Media, LLC 2011
Chapter 2
Proteomics of Epithelial Lining Fluid Obtained
by Bronchoscopic Microprobe Sampling
Lorenza Franciosi, Natalia Govorukhina, Nick ten Hacken,
Dirkje Postma, and Rainer Bischoff
Abstract
Epithelial lining fluid (ELF) forms a thin fluid layer that covers the mucosa of the alveoli, the small airways,
and the large airways. Since it constitutes the first barrier between the lung and the outer world, it is an
interesting target for proteomics studies that focus on lung disease. Bronchoscopic microprobe (BMP)
sampling of ELF uses small probes with an absorptive tip that are introduced bronchoscopically.
In contrast to other methods used so far for the collection of biofluids from the lung (e.g., broncho-
alveolar lavage fluid, induced sputum), this technique has the advantage that ELF is not diluted and
contains high concentrations of biomolecules. In addition, the investigated location in the tracheobron-
chial tree is well defined, and there is no contamination with oropharyngeal bacteria or saliva. Despite
occasional blood contamination of the probes by scratching the mucosa of the airways, the proteomic
analysis of microprobe-sampled ELF opens new possibilities for research in lung diseases. Our work
focuses particularly on the induction and progression of cigarette smoke-induced Chronic Obstructive
Pulmonary Disease (COPD).
In this chapter, we describe the practical aspects of sampling ELF followed by a detailed description
of proteomics analysis by LC-MS/MS after protein separation by SDS-PAGE and in-gel digestion. As an
example, we apply this proteomic platform to the identification and quantification of proteins in ELF from
COPD patients and healthy subjects.
Key words: Epithelial lining fluid, Bronchoscopic microsampling probe, Proteomics, 1D-SDS-PAGE
electrophoresis, Mass spectrometry
Increasingly, proteomics is an important tool in the discovery of
biomarkers for human diseases. Proteomics studies focusing on
lung diseases have used resected lung tissue, bronchial biopsies,
bronchoalveolar lavage fluid (BALF), induced sputum, exhaled
1. Introduction

18 L. Franciosi et al.
breath condensate, nasal lavage fluid, serum, urine, and cultured
cells. In the field of Chronic Obstructive Pulmonary Disease
(COPD), proteomic studies have used biopsies, BALF, and sputum
to detect approximately 40 candidate proteins that may play a role
in the pathogenesis of COPD (1–6). These proteins have different
functions in tissue repair and proliferation, the immunological
response and inflammation, cytoskeletal function, and protection
against oxidants. However, none of these proteins have been validated
in COPD (7).
Epithelial lining fluid (ELF) forms a thin aqueous layer that
covers the inner part of the airways and protects them against a
hostile outer world. This first barrier of innate defense is, therefore,
an interesting focus of research, due to the fact that allergens,
microbial agents, and toxic agents from cigarette smoke and air
pollution are responsible for lung diseases such as atopic asthma,
pneumonia, or COPD. ELF contains cells and soluble components
that probably play an essential role in this first line of defense.
Consequently, it has been proposed that the protein composition
of ELF will reflect the effects of external factors that affect the
lung, and that detection of changes in the ELF proteome will
be useful to diagnose, characterize, and potentially prognosticate
the pathological processes that are related to the progression of
lung disorders (8).
Proteomics studies on ELF have most frequently used BALF.
However, this way of collecting ELF has a number of disadvan-
tages as follows: (1) BALF dilutes the ELF by a factor of 60–120-
fold (9), which decreases the sensitivity to detect proteins that are
not abundantly present in the bronchial tree, and the dilution of
ELF may vary from one lavage to the next with no accepted method
to correct for this variation; (2) the exact location in the bronchial
tree from which proteins are derived is not known, because BALF
is collected from the fourth generation of tracheobronchial tree;
(3) there is a close relationship between certain proteins in serum
and BALF (5, 6), which poses questions about the lung specificity
of the BALF proteome. Analysis of induced sputum might be an
alternative to ELF or BALF analysis, since it can be obtained in a
noninvasive way. However, the area from which induced sputum is
collected is even more variable than that of BALF. Additionally,
induced sputum passes the oropharyngeal cavity, which contains
many bacteria and saliva that may easily contaminate sputum sam-
ples. Finally, dithiothreitol (DTT) is necessary to liquefy the highly
viscous sputum, which may in turn affect proteomics results.
Bronchoscopic microprobe (BMP) sampling of ELF is a prom-
ising technique in proteomics research of the lung. This technique
uses a small adsorptive pad that is placed on the mucosal layer of
the bronchial wall (second generation) under visual control during
bronchoscopy. It may also be placed in the smaller airways using

19
2 Proteomics of Epithelial Lining Fluid
radiography. In contrast to the collection of BALF or induced
sputum, the area from which ELF is obtained is precisely identified.
Additionally, there is no dilution of proteins, and the risk of con-
tamination with bacteria is minimal, since the probes are protected
by an extra sheath within the bronchoscope during entry and exit.
This collection technique has been used successfully in humans
with Acute Respiratory Distress Syndrome (ARDS) and small
peripheral lung carcinoma (10, 11), as well as, in studies measuring
drug concentrations in ELF. Microsampling probes were used to
collect ELF without the help of a bronchoscope in anesthetized,
ventilated rabbits that was followed by proteomic analysis that lead
to the identification of 43 proteins (12). More than 50% of these
proteins have been reported earlier in relation to lung cancer, lung
inflammation, and ARDS. Proteomics of microprobe-sampled
ELF opens a new area of research in lung disease, particularly in
those diseases with a strong inflammatory response to inhaled
agents. In that perspective, the response to cigarette smoke and the
possible induction of COPD is a particularly promising field for
ELF proteomics.
In this chapter, we describe a methodology for performing
proteomics in microprobe-sampled ELF. We initially describe how
ELF is sampled, which is followed by the extraction of proteins
from the probe, sample preparation, and protein separation by
SDS-PAGE with subsequent in-gel digestion of excised protein
bands. The identification of proteins, by LC-MS/MS of the digests
and database search, provides an overview of the ELF proteome.
To our knowledge, no proteomics studies have been performed in
human ELF. Therefore, our platform opens up new avenues for a
better understanding of the onset and progression of COPD and
possibly other lung disorders with implications for diagnosis and
prognosis, as well as, for drug discovery and development.
1. Bronchoscopic microsampling probes, BC-401C (Olympus
Corp., Tokyo, Japan) (see Fig. 1), BC-401C dimensions: working
length: 1,050 mm, fiber rod diameter: 1.1 mm, fiber rod
length: 30 mm, maximum insertion portion diameter: 1.8 mm.
These probes need a bronchoscopic working channel with an
inner diameter of at least 2.0 mm.
2. PBS buffer (sterile): 140 mM NaCl, 9 mM Na2
HPO4
, 1.3 mM
NaH2
PO4
.
3. Micro BCA Assay kit (Pierce Protein Research Product,
Thermo Scientific, Rockford, IL).
2. Materials
2.1. Microsampling
Probes and ELF
Extraction and Protein
Extraction

All the buffers are prepared with ultrapure water (18.2 Mů cm).
1. Loading buffer: 10% SDS, 10 mM DDT, 20% glycerol, 0.2 M
Tris–HCl, pH 6.8, 0.05% bromophenol blue.
2. Separating buffer: 1.5 M Tris–HCl, pH 8.8, 0.4% SDS (w/v).
Store at room temperature for up to 6 months (see Note 1).
3. Stacking buffer (4×): 0.5 M Tris–HCl pH 6.8, 0.4% SDS (w/v).
Store at room temperature for up to 6 months (see Note 2).
4. Acrylamide/bisacrylamide solution: 30% acrylamide/Bis solu-
tion (BioRad, Veenendal, The Netherlands) 37.5:1. Store for
up to 1 year at 4ºC according to the manufacturer’s
recommendations.
5. N,N,N,Nc-Tetramethyl-ethylenediamine (TEMED, BioRad).
Store at room temperature in the dark for up to 6 months.
6. APS (ammonium persulfate) (BioRad): stock solution of 10%
(w/v) in water in 100 PL aliquots. Store at −20ºC until needed.
7. Dissolve running buffer (10×): 0.25 M Tris–HCl, 1% SDS, 2 M
glycine, pH 8.3 in 1 L ultrapure water (18.2 Mů cm). Store at
room temperature for up to 3 months. For 1× concentrated solu-
tion, dilute 100 mL in 1 L of ultrapure water (18.2 Mů cm).
8. PageRuler™ Prestained Protein Ladder (Fermentas, St. Leon-Rot,
Germany) is used as molecular weight marker.
9. SDS-PAGE equipment: Mini-Protean III electrophoresis
system (BioRad).
1. Staining: 1 g Coomassie Brilliant Blue R concentrate, 90 mL
of acetic acid, 250 mL ethanol.
2. Destaining: 5% of acetic acid, 50% of methanol or ethanol, 45%
of ultrapure water.
1. Fixing solution: acetic acid:methanol:ultrapure water in a ratio
of 1:4:5 (see Note 3).
2. Sensitization solution: 75 mL of methanol, 10 mL of sodium
thiosulfate 5%, 17 g sodium acetate, 165 mL of ultrapure water.
2.2. SDS-PAGE
2.3. Coomassie Blue
Staining and
Destaining
2.4. Silver Staining
Fig. 1. Representation of a Bronchoscopic Microsampling Probe (BC-401C, Olympus,Tokyo,
Japan; ﬁber rod diameter: 1.1 mm, ﬁber rod length: 30 mm). Courtesy of Olympus,
Tokyo, Japan.

21
3. Staining solution: 0.6 g silver nitrate, 250 mL of ultrapure
water.
4. Developing solution: 6.25 g sodium carbonate, 100 PL form-
aldehyde, 250 mL of ultrapure water.
5. Stop solution: 3.65 g EDTA, 250 mL of ultrapure water.
1. Silver destaining: 30 mM potassium ferricyanide and 100 mM
sodium thiosulfate, protect from light. Store at 4°C until
needed.
2. Coomassie destaining: 50 mM NH4
HCO3
/50% acetonitrile.
3. 10 mM DDT solution: 1.54 mg DDT in 1 mL of 50 mM
NH4
HCO3
.
4. 55 mM iodoacetamide solution: 10 mg iodoacetamide in 1 mL
of 50 mM NH4
HCO3
.
5. Enzyme: 10 Pg/mL trypsin (sequencing grade modified
trypsin) (Promega, Madison, WI).
6. Trypsin buffer: resuspension buffer is provided in the kit of
sequencing grade modified trypsin (Promega).
1. Solvent A1 nano pump ion trap: ultrapure water, 0.1% of
formic acid.
2. Solvent B1 nano pump ion trap: 100% of acetonitrile, 0.1% of
formic acid.
3. Solvent A capillary pump ion trap: ultrapure water, 3% of ace-
tonitrile, 0.1% of formic acid.
4. LC-MS/MS equipment (Agilent Technologies, Santa Clara,
CA) as follows: autosampler; solvent degasser; nanopump;
capillary loading pump; chip cube interface.
1. Perform bronchoscopy using established guidelines (13).
Patients are not allowed to drink or eat 5 h prior to bronchos-
copy. On arrival, give patients 40 Pg ipratropium bromide via
spacer.
2. Then, 15 min later, instill 2 mL of 2% lidocaine in the mouth
(3–5×), the oropharynx, and on the vocal cords, as well as, into
the trachea to inhibit coughing. The total lidocaine dose is not
allowed to exceed 4.5 mg/kg (14).
3. Introduce a flexible bronchoscope with a minimum diameter
of the working channel of 2.0 mm into the left main bronchus.
Sample ELF by advancing the microsample probe, BC-401C
2.5. In-Gel Digestion
2.6. LC Solvents
3. Methods
3.1. ELF Collection
with Bronchoscopic
Microsampling Probe

(Olympus, Tokyo, Japan), into the lumen, and make gentle
contact with the mucosal layer. Ask the patient to hold his/her
breath for 10 s to avoid friction and thus, possible bleeding.
Discard probes with visual blood contamination.
1. Cut the collected adsorptive tips of each microprobe about
3 cm length and insert into 1.5 mL Eppendorf tubes (Reaction
tubes GmbH, Greiner Bio-One, Monroe, NC). Store on ice
containing 10 PL of PBS each.
2. Centrifuge tubes for 5 min at 2,000×g at 4°C, and remove the
probe. Bring liquid inside the tube up to 250 PL with PBS and
insert into a new tube containing 1 mL of PBS, and store on ice.
3. Rotate tubes for 10 min at 4°C to extract proteins. Remove
probes with the help of tweezers, and centrifuge the extracts
(Eppendorf Centrifuge, USA Scientific, Inc., Ocala, FL) for
5 min at 2,000×g at 4°C to remove insoluble material.
4. Insert the probe into a new Eppendorf tube containing 1 mL
of PBS, and repeat step 3.
5. Hereafter, insert the probe into a new empty Eppendorf tube,
and centrifuge under the same conditions, as described in
steps 3 and 4 (see Subheading 3.2) to recover the adsorbed
liquid. Finally, discard the absorptive tip, and pool the extracts
(i.e., total volume: 2.25 mL), aliquot, and store at −80°C until
needed.
1. Determine the total protein concentration in Costar transparent
96-well plates (Corning, Schiphol-Rijk, The Netherlands)
using the Micro BCA Assay (Pierce Protein Research Product,
Thermo Scientific, Rockford, IL), according to the manufac-
turer’s protocol.
2. Using a plate reader (Molecular Devices, THERMOmax,
Scientific Support, Hayward, CA), read absorbance at 550 nm.
1. Perform SDS-PAGE in 1.0-mm thick, 12.5% polyacrylamide
gels in a Mini-Protean III electrophoresis system (BioRad) at
20 mA.
2. Prepare 12.5% polyacrylamide separating gel by mixing
1.25 mL of separating buffer, 2.1 mL of acrylamide/bis (30%)
stock solution, 1.6 mL of water, 50 PL of APS (10%), and 2 PL
of TEMED.
3. Pour the gel between clean glass plates that were washed in
the following order: ultrapure water, 0.1% SDS added to ultra-
pure water, ultrapure water, ethanol, and then, dry with clean
paper.
4. Overlay the separating gel with isopropanol until polymeriza-
tion (approximately 30–40 min). Then, remove isopropanol,
3.2. Sample
Preparation
3.3. Determination of
Protein Concentration
3.4. SDS-PAGE

23
and extensively wash the top of the separating gel with ultra-
pure water and dry with filter paper.
5. Prepare stacking gel by initiating polymerization of 1.43 mL of
water, 0.42 mL of acrylamide/bis (30%) stock solution, and
0.62 mL of stacking buffer through an addition of 25 PL of APS
(10%) and 2.5 PL TEMED after insertion of the appropriate
combs.
6. After approximately 30 min, remove the combs, and wash the
wells 1× with running buffer.
7. Assemble the cassettes as follows: fill the inside chamber (cath-
ode chamber) and the outside chamber (anode chamber) with
running buffer 1× to completely cover the top and the bottom
of the gel. Run the gel with a starting voltage of 120 V and
increase to 180 V until the tracking dye reaches the bottom of
the gel (see Note 4).
1. Staining with Coomassie Brilliant Blue to reveal proteins: stain
the gel in a plastic or glass box with Coomassie Brilliant Blue R
concentrate 1 g in 90 mL of acetic acid and 250 mL of ethanol
on a shaker for 2 h (see Note 5). Subsequently, replace the
staining solution with the destaining solution as follows: 5% of
acetic acid, 50% of methanol or ethanol, and 45% of ultrapure
water to clear the gel from the background color and to visualize
the bands corresponding to the proteins.
2. Silver staining (see Note 6): as soon as the electrophoretic run is
finished, transfer the gel to either a glass or plastic box contain-
ing the fixing solution: acetic acid:methanol:ultrapure water in a
ratio of 1:4:5 for 15 min. Then, remove the solution, and add
a fresh fixing solution, leaving the gel for an additional 15 min.
3. After step 2, submerge the gel for 30 min in sensitization
solution: 75 mL of methanol, 10 mL of 5% sodium thiosul-
fate, 17 g sodium acetate, 165 mL of ultrapure water, and
wash 3× for 5 min each with ultrapure water.
4. Add staining solution: 0.6 g silver nitrate, 250 mL of ultrapure
water, freshly prepared, and leave the gel for 20 min on a
shaker. After washing 2× for 1 min each with ultrapure water,
pour the developing solution over the gel (see Note 7) until
the bands become visible. Finally, discard this solution and
replace by the stop solution (see Note 8 and Fig. 2) (15).
1. Cut the bands of interest out of the gel (see Note 9) with the
help of a clean surgical blade (see Note 10), and place each
band into a clean Eppendorf tube. In the case of silver staining,
add 200 PL of the silver destaining solution, 3.65 g EDTA,
250 mL of ultrapure water to the Eppendorf tube containing
the gel piece, and incubate for 20 min in the dark.
3.5. Gel Staining
3.6. In-Gel Digestion

2. Remove the supernatant, and add 100 PL of ultrapure water,
and leave for 15 min with periodical vortexing. Remove the
supernatant, and repeat the washing with ultrapure water until
the gel pieces are clear (16).
3. For Coomassie-stained gel pieces, add 100 PL of the destaining
solution, 5% of acetic acid, 50% of methanol or ethanol, 45% of
ultrapure water to the tubes, and leave for 10 min. Centrifuge
for 10–15 s at 1,000×g, then, discard the supernatant. Repeat
step 3 until the gel pieces are colorless (see Note 11).
4. At this point, add 100 PL of 100% acetonitrile to the gel pieces,
incubate for 5 min, and vortex 2×. Centrifuge for 10–15 s at
1,000×g and discard the supernatant. Repeat step 4 until the
gel pieces appear white and shrunken.
5. Cover the gel pieces with 100 PL of 100 mM DDT and incubate
at 50°C for 30 min.
6. Centrifuge for 10–15 s at 1,000×g and remove the liquid,
then, add 100 PL of acetonitrile to shrink the gel, and remove
the excess liquid. Cover the shrunken gel pieces with 100 PL
of 55 mM iodoacetamide, and incubate at room temperature
for 1 h in the dark while wrapping the Eppendorf tube with
aluminum foil (see Note 12).
7. Centrifuge the tubes for 15 s at 1,000×g, discard the liquid,
and wash the gel pieces with 200 PL of 50 mM NH4
HCO3
for
Fig. 2. SDS-PAGE analysis of proteins in Epithelial Lining Fluid (ELF) obtained after
Bronchoscopic Microprobe Sampling and detected by silver staining. Five different probes
from ﬁve different patients are analyzed. Right lane: molecular weight marker (kDa).
Franciosi et al., unpublished data.

25
15 min with 2× vortexing. After centrifugation and removal of
the liquid, add 100 PL of 100% acetonitrile, and leave the gel
pieces for 5 min. Remove excess liquid, and keep the tubes open
in order to let the gel pieces dry under the hood for about 5 h
(see Note 13).
8. Prepare the solution of trypsin for the digestion as follows:
remove 20 Pg vial of trypsin from the freezer and add 100 PL
of trypsin buffer [10 Pg/mL trypsin (sequencing grade
modified trypsin, Promega, Madison, WI) to the tube. After
thorough mixing, prepare aliquots of 8 PL and add 136 PL of
50 mM NH4
HCO3
, and mix well. Store the other aliquots in
the freezer until further use.
9. Add 20 PL of trypsin solution to each dried gel piece and
rehydrate at room temperature for 1 h. In the case that the gel
piece is not well covered by the trypsin solution, add additional
50 mM NH4
HCO3
(see Note 14). Perform digestion at 37°C
overnight. To stop the reaction, add 0.75 PL of formic acid the
following day.
10. At this point, transfer samples (about 15 PL each) to brown
screw-cap vials (Agilent Technologies, Amstelveen, The
Netherlands) with 300 PL point inserts with a polyspring (Waters,
Etten-Leur, The Netherlands) for LC-MS/MS analysis.
1. Identify proteins after in-gel digestion by nano-LC-MS/MS
analysis using a microfluidics chip-cube interface that includes
a chip with a 40 nL enrichment column of 75 Pm×11 mm and
a 75 Pm×150 mm separation column packed with Zorbax
300SB-C18 (Agilent Technologies), 5 Pm particle diameter
chromatographic material.
2. The interface contains a nanoelectrospray tip of 2 mm length
with conical shape: 100 Pm OD × 8 Pm ID coupled on-line
to an ion-trap mass spectrometer (MSD-Trap-SL, Agilent
Technologies).
3. Perform injections of each sample (3 PL) (see Note 15) with
an autosampler that is equipped with an injection loop of 8 PL
and a thermostated cooler that maintains the samples in the
autosampler at 4°C during analysis.
4. The chip-LC-MS/MS system contains the following additional
modules: nanopump, capillary loading pump, and a solvent
degasser.
5. Use two eluents for the nanopump: eluent A1, 0.1% formic
acid in ultrapure water and eluent B1, 0.1% formic acid in ace-
tonitrile. For the capillary pump to load the sample onto the
trap column, use one solvent: 0.1% formic acid, 3% acetonitrile
in ultrapure water. Perform sample loading at a flow rate of
3 PL/min.
3.7. LC-MS/MS

6. Perform elution for 5 min at a flow rate of 0.25 PL/min with
3% of eluent B1, a linear gradient from 3 to 53% in 57 min of
B1, and follow by a step gradient from 53 to 90% in 5 min of
eluent B1. Maintain 90% of eluent B1 for 10 min.
7. Use the following parameters for acquisition of MS/MS spectra
(these parameters are indicative and have to be adjusted for each
instrument): drying gas (N2
): 4.0 L/min, drying gas tempera-
ture: 300°C, skimmer: 40.0 V, cap. exit: 200.0 V, Oct1: 12.0 V,
Oct2: 2.50 V, Oct RF: 200.0 Vpp, Trap drive: 78.0, Lens 1:
−5.0 V, Lens 2: −60.0 V; polarity: positive, maximal accumulation
time: 15.00 ms, scan from 340 to 2,200m/z, averages: 4, target
mass: 622.0m/z. Analyze the original MS/MS spectra with the
Bruker Daltonics Data Analysis software version 3.4. (Bruker
Daltonics, Bremen, Germany). Automatically select 300 com-
pounds in a retention time window between 18 and 75 min and
deconvolute with respect to charge state and isotopes.
8. For the identification of the proteins, export MS/MS spectra as
Mascot generic files (Matrix Science, London, UK) and submit
to a web-based version (http:/
/www.matrixscience.com) of
Mascot (version 2.2) to query the Swissprot and UniProt data-
bases. The search parameters in Mascot are listed (see Table 1).
9. Follow the HUPO guidelines for criteria for acceptance, which
stipulate that proteins must be identified with at least two or
more significant peptides and that the false discovery rate that
is based on a decoy database should be lower than the 5%.
Table 1
Search parameters used in Mascot
Taxonomy Homo sapiens
Database Swissprot 56.5
Peptide charge 1+, 2+, 3+
Variable modifications Carbamidomethyl (C)
Oxidation (M)
Quantitation None
Enzyme Trypsin
Allow up to 3 missed cleavages
Peptide tolerance ±1.6 Da
MS/MS tolerance ±0.8 Da
Data format Mascot generic
Instrument ESI-TRAP
Decoy Enabled

27
1. Dissolve the components in 80 mL of ultrapure water
(18.2 Mů cm), adjust the pH to 8.8, and then fill up to 100 mL
with ultrapure water.
2. Dissolve the components in 80 mL of ultrapure water
(18.2 Mů cm), adjust the pH to 6.8, and then fill up to 100 mL
with ultrapure water.
3. Fixing and sensitization solutions can be prepared in advance,
but staining, developing, and stop solutions must be prepared
just prior to being used.
4. The voltage is kept at 120 V until the tracking dye reaches the
bottom of the stacking gel and then, is increased to 180 V until
the end of the run.
5. Also, the gel can be stained overnight, but in that case, destaining
will take longer.
6. Silver staining is preferred when more sensitive detection is
required.
7. In order to avoid too much background staining, it is advisable
to change the developing solution 2–3× and continue to shake
manually until visualization of the bands.
8. The gel can be preserved in this solution at 4°C for months.
9. In order to cover the gel pieces with liquid and have a better
adsorption of the enzyme for digestion, the gel pieces must not
be too big. If that is the case, just cut them into smaller pieces
with the help of the blade or a pipette tip.
10. After cutting one band, clean the blade with ultrapure water.
When cutting bands from different samples (i.e., different
lanes), clean the blade with ethanol and subsequently with
ultrapure water in order to avoid contamination.
11. If you have to interrupt the procedure, it is best to do so after
destaining. Add 100 PL of ultrapure water to each gel piece,
and store at 4°C until the next day.
12. It is important to cover the Eppendorf tubes containing the
shrunken gel pieces in this solution with foil, since iodoacet-
amide is light-sensitive.
13. It is important to keep in mind that the dried gel pieces at this
point are smaller and electrostatic. They can jump out of the
tubes easily.
14. Adding 5–8 PL of 50 mM NH4
HCO3
is generally sufficient.
15. It is recommended to intersperse blank runs in the analysis
sequence of the samples (i.e., usually one blank run of 50:50%
ultrapure water:acetonitrile, after every 5–8 sample runs) to
avoid and check for carry over.
4. Notes

Acknowledgments
The authors wish to acknowledge their appreciation to the Olympus
Medical Systems Cooperation for providing the microsampling
probes. The work presented in this chapter is financially supported
by the Top Institute Pharma (project number: T1-108).
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