Spectral Techniques In Proteomics

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SPECTRAL TECHNIQUES IN PROTEOMICS

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SPECTRAL TECHNIQUES IN PROTEOMICS Edited by

DANIEL S. SEM

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

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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 consequences 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 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

2006103310

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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.

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Table of Contents Preface....................................................................................................................... xi Editor ......................................................................................................................xiii Contributors ............................................................................................................. xv Abbreviations .......................................................................................................... xix

PART I Chapter 1

The Scope of Proteomic and Chemical Proteomic Studies 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 Chapter 4

Mass Spectral Studies of Proteome and Subproteome Mixtures 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 vii

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Chapter 7

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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

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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

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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 comprehensive 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 innovation, 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 interactions; (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 xi

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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 identification. 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 techniques 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

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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 development 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, cheminformatic 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.

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Contributors Alex Aronov Vertex Pharmaceuticals Incorporated Cambridge, Massachusetts

Eric Fung Ciphergen Biosystems Inc. Fremont, California

Peter E. Barker Biotechnology Division National Institute of Standards and Technology Gaithersburg, Maryland

Michael B. Goshe Department of Molecular and Structural Biochemistry North Carolina State University Raleigh, North Carolina

Guy Bemis Vertex Pharmaceuticals Incorporated Cambridge, Massachusetts

Per Hägglund Biochemistry and Nutrition Group Technical University of Denmark Lyngby, Denmark

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

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

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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

Contributors

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

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Contributors

Matthias Pelzing Bruker Daltonik GmbH Leipzig, Germany

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Al Pierce Vertex Pharmaceuticals Incorporated Cambridge, Massachusetts

Jeremy Spater Departments of Radiology and Bioengineering University of Pittsburgh Cancer Institute MR Research Center University of Pittsburgh Pittsburgh, Pennsylvania

Anthony W. Purcell Department of Biochemistry and Molecular Biology Bio21 Molecular Science and Biotechnology Institute University of Melbourne Victoria, Australia

Sunitha B. Thakur Departments of Radiology and Bioengineering University of Pittsburgh Cancer Institute MR Research Center University of Pittsburgh Pittsburgh, Pennsylvania

Rebecca L. Rich Center for Biomolecular Interaction Analysis School of Medicine University of Utah Salt Lake City, Utah

Sakanyan Vehary ProtNeteomix Université de Nantes Nantes, France

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

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

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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

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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 xix

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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

Abbreviations

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Abbreviations

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

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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

Abbreviations

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Part I The Scope of Proteomic and Chemical Proteomic Studies

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The Systems-Based Approach to Proteomics and Chemical Proteomics Daniel S. Sem

CONTENTS 1.1 1.2 1.3

Introduction ...................................................................................................... 3 Complexity and Dynamic Range Challenges.................................................. 4 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) technologies. 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. 3

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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 modifications). 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