Clinical Proteomics

From Diagnosis to Therapy

Edited by

Jennifer E. Van Eyk and Michael J. Dunn

InnodataFile Attachment9783527622160.jpg

Clinical Proteomics

Edited by

Jennifer E. Van Eyk and

Michael J. Dunn

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Clinical Proteomics

From Diagnosis to Therapy

Edited by

Jennifer E. Van Eyk and Michael J. Dunn

The Editors

Dr. Jennifer van Eyk

Johns Hopkins University

Bayview Medical Campus

5200 Eastern Ave.

Baltimore, MD 21224

USA

Prof. Dr. Michael J. Dunn

Proteome Research Centre

UCD Conway Institute

Belfield

Dublin 4

Ireland

Cover

The front cover picture is based on a 2-D-DIGE

comparing a cell lysate preparation and secretome

preparations, both derived from SW620 cells. It was

published by Schwarte-Waldhoff et al. in Proteomics

Clin. Appl. 2007, 1, 47-61. Lysate proteins are shown

in red and secretome proteins in green.

Reproduced with kindly permission of the Medical

Proteome-Center, University of Bochum, Germany.

The background protein image has been reproduced

with the kind permission of Professor Alfredo Ricci,

Dpto. di Chimica Organica, Universita di Bologna,

Italy.

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Contents

Editor’s Overview XIX

List of Contributors XXI

I Technologies 1

1 Preanalytical Issues in Clinical Proteomic Studies 3Roisean E. Ferguson, Rosamonde E. Banks

1.1 Introduction 31.2 Preanalytical Factors 31.2.1 Biological Variation 41.2.1.1 Intrinic Influences/Factors 41.2.1.2 Extrinsic Influences/Factors 51.2.2 Technical Variables 51.2.2.1 Specimen/Sample Collection Mode 51.2.2.2 Type of Sample Container 61.2.2.3 Sample Processing and Handling Conditions 71.2.2.4 Sample Storage 71.3 Summary and Concluding Remarks 8

2 Protein Separation by Two-Dimensional Electrophoresis 13Pamela M. Donoghue, Miroslava Stastna, Michael J. Dunn

2.1 Introduction 132.2 2DE: Protein Solubilization and Sample Preparation 142.3 2DE: Protein Separation 152.3.1 Focusing in the First Dimension 152.3.2 Advances in IEF 162.4 Improving Proteomic Coverage by Subcellular Fractionation 212.5 Protein Detection and Image Analysis 242.6 The Future of 2DE 25

V

3 Protein Separation: Liquid Chromatography 31Miroslava Stastna, Jennifer Van Eyk

3.1 Introduction 313.2 Liquid Chromatography 323.2.1 HPLC Separation Principles 323.2.2 Reversed-Phase LC (RPLC, 1DLC) 343.2.3 Affinity Chromatography 373.2.4 Size-Exclusion Chromatography 383.2.5 Ion-Exchange Chromatography 393.2.6 Two-Dimensional LC 403.2.6.1 Chromatofocusing to Reversed Phase 403.2.6.2 Ion-Exchange Reversed-Phase Liquid Chromatography 413.2.7 Three-Dimensional Liquid Chromatography 413.2.8 LC Image Analysis Requirement 423.2.9 Mass Spectrometry for LC 423.2.9.1 MALDI-TOF MS 433.2.9.2 ESI-MS/MS 433.3 Conclusions 46

4 HPLC in Protein Discovery 53Timothy J. Barder

4.1 Introduction 534.2 LC-Based Approaches in Peptide Mass Mapping 544.3 LC-Based Approaches in Protein Mapping 544.4 Orthogonal 2D HPLC Separations 564.5 Conclusion 57

5 IEF Analysis of Peptides for Biomarkers Discovery 61Ali R. Vaezzadeh, Catherine G. Zimmermann-Ivol, Jacques Deshusses,

Alexander Scherl, Denis F. Hochstrasser

5.1 Introduction 615.2 Background 625.2.1 Isoelectric Focusing 625.2.2 Shotgun Proteomics 625.2.3 Shotgun IEF 635.3 Shotgun IEF Workflow 645.4 Applications 665.5 Discussion and Outlook 66

6 Capillary Electrophoretic Separations for Clinical Proteomics 73Brian M. Balgley, Weijie Wang, Xueping Fang, Don L. DeVoe, Cheng S. Lee

6.1 Introduction 736.2 (Single-Dimension) Capillary Electophoretic Separation 746.3 Capillary Electrophoresis-Based Multidimensional Separations 746.3.1 Capillary Liquid Chromatography–Capillary Electrophoresis 75

VI Contents

6.3.2 Capillary Electrophoresis–Capillary Electrophoresis 756.3.3 Capillary Electrophoresis–Liquid Chromatography 766.3.3.1 Characterization of Human Saliva Proteome 776.3.3.2 Targeted Tissue Proteomics 796.4 Conclusions 85

7 Quantitative Proteomics Using Nano-LC with High Accuracy

Mass Spectrometry 89Ljiljana Paša-Tolić, Jon M. Jacobs, Wei-Jun Qian, Richard D. Smith

7.1 Introduction 897.2 Fundamentals of a High Mass Accuracy-Based LC–MS

Approach 907.3 Nano-LC–MS for Enhanced Sensitivity and Dynamic

Range Coverage 927.4 Further Developments for Increasing Proteomic Throughput 947.5 Obtaining More Robust Quantitative Proteomic Measurements 947.6 Summary and Perspective 96

8 Antibody Microarrays for Protein and Glycan Detection 101Songming Chen, Brian B. Haab

8.1 Introduction 1018.2 Antibody Preparation and Microarray Production 1028.3 Sandwich Assays with Fluorescence Detection 1048.4 Antibody Microarrays with Lectin Detection 1068.5 Conclusion 1068.6 Detailed Protocols 107

9 Biomarker Identification: The Role of Experimental Design,

Statistics, and Data Sharing 113Marc R.Wilkins

9.1 Introduction 1139.2 Experimental Designs for Biomarker Discovery 1149.3 Identification of Biomarker Proteins 1169.4 Biomarker Validation and the Issue of Data Sharing 1189.5 Conclusions 119

II Cancer 121

10 Applications of Stable Isotope Tagging Based Quantitative

Proteomics in Cancer Research 123Ru Chen, Teresa A. Brentnall, Ruedi Aebersold

10.1 Introduction 12310.2 Stable Isotope Tagging Methods 12410.2.1 Chemical Labeling of Stable Isotope Tags 125

Contents VII

10.2.2 Biological Incorporation of Stable Isotope Tags 12710.3 Applications in Studies of Tissue Samples 12810.3.1 Whole Tumor Tissue Labeled with ICAT 12810.3.2 Whole Tumor Tissue Labeled with ICAT and iTRAQ 13010.3.3 Isolated Tumor Cells Labeled with ICAT 13110.3.4 Isolated Tumor Cells Labeled with 16O/18O 13110.4 Applications in Studies of Bodily Fluids 13210.4.1 Pancreatic Juice Labeled with ICAT 13310.4.2 Nipple Aspirate Fluid Labeled with ICAT 13310.4.3 CSF 13410.5 Applications in Studies of Cell Lines 13510.5.1 Ovarian Cancer Cell Lines Labeled with ICAT 13510.5.2 Breast Cancer Cell Lines Labeled with 18O Labeling 13510.5.3 Prostate Cancer Cell Lines Labeled with SILAC 13610.5.4 Secretome by Pancreatic Cancer Cell Line Labeled with SILAC 13610.6 Applications in Studies of Protein Interaction 13710.7 Applications in Studies of Posttranslational Modifications (PTM) 13810.8 Summary 139

11 Two-Dimensional Liquid Separations, Protein Microarrays,

and Mass Spectrometry in Comprehensive Analysis of

Posttranslational Modifications and Biomarker

Discovery in Cancers 145Tasneem H. Patwa, Jia Zhao, David E. Misek, David M. Lubman

11.1 Challenges in Biomarker Discovery: The Emerging Role of

Posttranslational Modifications 14511.2 Proteomics in Disease Research 14611.3 The Problem of Identifying and Characterizing Posttranslational

Modifications: Current Efforts 14711.4 Microarrays in Proteomic Investigations 14811.5 A Comprehensive Method Combining Liquid Separations,

Microarrays, and Mass Spectrometry 14811.6 2D Liquid-Based Separations and Mass Mapping 15011.7 Posttranslational Modification (PTM) Analysis 15211.8 Phosphorylation 15311.9 Glycosylation 15411.10 Autoimmune (Humoral) Response Studies 15611.11 Future of a 2DLC, Microarray Methodologies in Discovery and

Diagnostics 158

12 Development and Use of Reversed-Phase Protein Microarrays

for Clinical Applications 165Virginia Espina, Julia Wulfkuhle, Valerie S. Calvert,

Kirsten H. Edmiston, Lance A. Liotta, Emanuel F. Petricoin III

12.1 Introduction 165

VIII Contents

12.2 The Growing Role of Protein Arrays in Molecular Diagnostics 16712.3 Reversed-Phase Arrays: Enabling Technology for Patient-Tailored

Therapeutics 16712.4 Use of Reversed-Phase Arrays for Signal Pathway Profiling

of Human Cancer 16912.5 Use of Reversed-Phase Arrays: A View to the Future 171

13 Cyclin-Dependent Kinase Inhibitors and Cancer: Usefulness

of Proteomic Approaches in Assessment of the Molecular

Mechanisms and Efficacy of Novel Therapeutics 177Marian Hajduch, Helena Skalnikova, Petr Halada, David Vydra,

Petr Dzubak, Marta Dziechciarkova, Miroslav Strnad,

Danuta Radioch, Suresh Jivan Gadher, Hana Kovarova

13.1 Introduction 17713.2 Proteomic Analysis of Cancer Cells Responding to the

Synthetic CDKI 18213.3 Two-Dimensional Protein Maps of Cancer Cells Treated by CDKI 18413.3.1 Model of Hematological Malignancy: CEM T-Lymphoblastic

Leukemia 18413.3.2 Solid Tumor Model: A549 Lung Adenocarcinoma Cells 18713.4 Evaluation of the Protein Maps: Possible Pathways Relevant to

Anticancer Effects of CDK Inhibition 18913.4.1 Candidate Biomarkers Identified Using the Hematological

Malignancy Model 18913.4.2 Candidate Biomarkers Identified Using the Solid Tumor Model 19213.5 Biomarker Validation Studies Focused on the crkl Protein 19413.6 Conclusions 195

III Cardiac Disease 203

14 Diagnostic Markers for Monitoring Heart Transplant Rejection 205Ciara A. McManus, Marlene L. Rose, Michael J. Dunn

14.1 Introduction 20514.2 Acute Rejection 20614.3 Chronic Rejection 20814.4 Cardiopulmonary Bypass 21214.5 Conclusions 213

15 The Study of Microheterogeneity in Human Plasma Proteins:

Application to Acute Myocardial Infarction 217Randall W. Nelson, Urban A. Kiernan, Dobrin Nedelkov,

Kemmons A. Tubbs, Eric E. Niederkofler

15.1 Background 21715.2 Technical Approach 218

Contents IX

15.3 Programmatic Study of Disease: Population Proteomics

Versus Myocardial Infarction 21915.3.1 Preliminary Screening, (Putative) Biomarker Discovery and

Identification 22215.3.2 Verification 22315.3.3 Knowledge Assembly and Next-Generation Assay Design 22315.3.4 Data Generation 22515.3.5 Data Analysis 22515.3.6 Blind and Randomized Challenge of Final Assay 22715.4 Summary 228

16 Discovery of Biomarkers for Cardiovascular Diseases 231Anthony O. Gramolini, Andrew Emili

16.1 Current Proteomic Technologies Available for CVD Biomarker

Searches 23216.2 Challenging Issues for Proteomic Profiling 23316.3 Screening Blood for Biomarkers 23416.4 Tissue Surveys 23516.5 The Value of Animal Models 23616.6 After Technology Platform and Sample Selection:

What Makes a Good Biomarker? 23716.7 Ongoing Considerations 23816.8 Outlook 238

17 Development of Biomarker Development Pipeline:

Search for Myocardial Ischemia Biomarkers 241Qin Fu, Shijun Sheng, Jennifer E. Van Eyk

17.1 Introduction 24117.2 Myocardial Ischemia and Infarction 24217.3 Lessons Learned from Cardiac Troponin 24517.4 Building a Biomarker Development Platform I-Discovery 24617.4.1 High-Abundant Protein Partitioning 24817.4.2 Utilizing Multiple Protein Separation Methods to Maximize

Proteome Coverage: A Synergistic Approach 25017.5 Validation 25417.5.1 Technologies in Validation 25617.5.2 Cohorts for the Validation 25617.6 Summary 257

18 The Albuminome as a Tool for Biomarker Discovery 263Rebekah L. Gundry, Robert J. Cotter

18.1 Protein–Protein Interactions and Protein-Centric

Approaches in Proteomics 263

X Contents

18.2 Defining the Albuminome 26518.3 The Albuminome as a Tool in Biomarker Discovery 27018.4 Role of the Albuminome in Cardiovascular Proteomics 27518.5 Other Plasma Subproteomes 27618.6 Conclusion 277

19 Application of Metabolomics to Cardiovascular

Biomarker and Pathway Discovery 279Gregory D. Lewis, Robert E. Gerszten

19.1 Introduction 27919.2 The Birth of Metabolomics 27919.3 Technologies to Define the Human Metabolome 28119.4 The Diagnostic Utility of Metabolic Peak Patterns:

A Call for Unambiguous Identification 28219.5 Pathway Analysis of Metabolomic Data 28319.6 Rationale for Metabolomic Approaches to Study Atherosclerosis

and its Complications: The Role of Proinflammatory Lipid

Metabolites 28519.7 Unanticipated Roles of ‘‘Intracellular’’ Metabolites 28619.8 Clinical Rationale for Applying Metabolomics to Coronary

Heart Disease 28619.9 Impediments to Human Applications 28719.10 Application of Metabolomics to Unique Human Cardiovascular

Disease Models 28719.11 Conclusion 290

IV Vascular Disease: Pulmonary, Diabetes and Brain 295

20 Urinary Biomarkers in Diabetic Nephropathy and Other

Glomerular Diseases 297John M. Arthur, T. Brian Powell

20.1 Urine as a Source of Protein Biomarkers 30020.2 Size Selectivity of Urine Proteins as a Marker 30120.3 Charge Specificity of Urine Proteins as a Marker 30220.4 Markers in Diabetic Nephropathy 30320.5 Markers for Other Glomerular Diseases 30420.6 Urine Proteomics 30420.7 Proteomics and Glomerular Disease Markers 30520.8 Diabetic Nephropathy 30820.9 Lupus Nephritis 31120.10 Other Glomerular Diseases 31220.11 Summary 315

Contents XI

21 Pulmonary Proteomics 323Jan Hirsch, Lorraine B. Ware, Michael A. Matthay

21.1 Introduction 32321.2 The Proteome of Bronchoalveolar Lavage Fluid 32321.3 BAL Studies in Animals 32521.4 Plasma and Serum Measurements 32621.5 Induced Sputum 32721.6 Pulmonary Edema Fluid 32821.7 Nasal Lavage Fluid 32921.8 Exhaled Breath Condensates 33021.9 Cell Analysis 33021.10 Frozen Tissue Slices 33521.11 Pleural Effusions 33721.12 Other Samples 33821.13 Summary 338

22 Proteomics Providing Insights into Major Psychiatric Disorders 349Melanie Föcking, Kyla Pennington, Jane English, Michael Dunn,

David Cotter

22.1 Introduction 34922.1.1 Schizophrenia and Affective Disorders: Definitions and

Epidemiology 35022.1.2 Schizophrenia and Affective Disorders: Brain Changes 35122.1.3 Effects of Psychiatric Drug Treatments on the Brain 35222.1.4 Application of Microarrays to Psychiatric Disorders 35222.1.5 The Value of Proteomic Approaches in Investigating the

Pathophysiology of Major Psychiatric Disorders 35322.1.6 Gel-Based Proteomic Methods 35422.1.7 Non-Gel-Based Methods 35522.1.8 Creating a Subproteome 35822.2 The Importance of Validation 35922.2.1 What Samples can be Used? 35922.3 Drug Discovery 36022.3.1 Pharmacoproteomic Investigations of the Brain 36022.4 Studies from Our Group 36122.4.1 Dorsolateral Prefrontal Cortex 36122.4.2 White Matter 36222.4.3 Anterior Cingulate Cortex 36322.4.4 Insular Cortex 36622.4.5 Hippocampus 36722.4.6 Studies of Effects of Psychotropic Medication 36822.4.7 Confounding Factors Influencing Human Postmortem Brain

Proteomic Studies 36922.4.8 Final Conclusions 369

XII Contents

V Toxicity, Bacterial and Viral Infections 379

23 Redox Proteomics Analysis of Oxidative Modified Brain

Proteins in Alzheimer’s Disease and Mild Cognitive Impairment:

Insights into the Progression of This Dementing Disorder 381Rukhsana Sultana, D. Allan Butterfield

23.1 Introduction 38123.1.1 Proteomics 38123.1.2 Mass Spectrometry and Database Searching 38423.1.3 Oxidative Stress in AD 38523.1.4 Oxidative Stress in MCI 38623.2 Redox Proteomics in AD and MCI 38723.2.1 Oxidized Proteins in AD and MCI Identified Using Redox

Proteomics 38823.2.1.1 Energy Dysfunction 38823.2.1.2 Proteasome-Related Proteins 38923.2.1.3 Cholinergic System 39023.2.1.4 Structural Proteins 39023.2.1.5 Cell Cycle, Phosphorylated Tau, and Ab Production 39123.2.1.6 pH Regulation Protein 39123.2.1.7 Neurotransmitter-Related Proteins 39123.3 Conclusion 392

24 Toxicoproteomics: Correlating Tissue and Serum Proteomics

in Liver Injury 403B. Alex Merrick

24.1 The Field of Toxicoproteomics 40324.2 Toxicoproteomics and Pharmaceutical Development 40424.3 Disciplines and Platforms for Toxicoproteomic Research 40624.4 Correlating Tissue and Serum Analysis 40824.5 Toxicoproteomic Studies in Liver Injury 41024.6 Acetaminophen 41124.7 Carbon Tetrachloride 41724.8 Bromobenzene 41824.9 Wyeth 14643 41824.10 Hydrazine 41924.11 Thioacetamide 42024.12 New Blood Biomarkers in Liver Injury 42124.13 Expectations and Reality 42324.14 Future Trends and Tools in Toxicoproteomics 42424.15 Summary 425

25 Biomarkers for Renal Disease and Uremic Toxins 435Eric Schiffer, Harald Mischak, Raymond C. Vanholder

25.1 Introduction 435

Contents XIII

25.2 Proteome Analysis 43625.3 Technical Aspect of CE–MS 43825.3.1 Ionization and Choice of Mass Spectrometers 43825.3.2 CE–MS Coupling 43925.3.3 Coating 44025.3.4 Sample Preparation 44025.3.5 Data Evaluation 44125.3.6 Identification of Biomarkers 44125.4 Application of Proteomic Techniques to Uremic Toxicity 44425.5 Conclusion 447

26 HIV and Other Viral Screens 453David R. Graham

26.1 Introduction 45326.2 Current State of Clinical Virology 45326.2.1 Diagnosis 45526.2.2 Treatment 45926.3 Predicting Pathogenicity: A Need for Clinical Viral

Proteomics 45926.4 Concepts in Virology as They Relate to Proteomics: The Problem

of Mutation 46026.5 Understanding the Biological Limitations of the Pathogen to

Succeed in a Proteomic Approach 46126.5.1 Culture 46126.5.2 Purity – Enveloped/Nonenveloped 46326.6 The Requirements and Approaches to Proteomic Research 46426.6.1 Decision Tree 46426.6.2 Broad-Based Approaches 46526.6.2.1 Protein Identifications or Posttranslational Modifications 46526.6.3 Liquid Chromatography-Based Approaches 46626.6.3.1 Reverse-Phase HPLC (rp-HPLC) – Separation Based on

Hydrophobicity 46726.6.3.2 Two-Dimensional HPLC 46826.7 Pushing the Envelope of Detection 46926.7.1 Protein Interactions 47026.7.1.1 Affinity Capture Based 47026.7.2 Protein Identification 47126.7.2.1 Antibody Based 47126.7.2.2 Mass Spectrometry Based 47126.8 Bioinformatics (Host Proteins Versus Viral Proteins) 47226.8.1 Are Molecular Approaches Sufficient? 47226.8.1.1 Laying the groundwork 47326.8.1.2 Virus sequencing 475

XIV Contents

26.8.2 Toward the Real World – Emerging Clinical Applications 47526.8.2.1 Protein Arrays 47526.8.2.2 The Evolution of Protein Arrays – Multiplex Assays 47626.8.3 Assessing Immune Responses Using Arrays and Multiplex

Assays 47626.8.3.1 Proper Data Analysis 47826.8.3.2 The Development of Clinical Cohorts for Validation 478

VI Autoimmune Disease and Autoantibodies 481

27 Application of Fungal Cyclic Peptides and Metabolites 483Jan Nedvěd, Miroslav Sulc, Alexandr Jegorov,

Anastassios Giannakopulos, Vladimir Havlicek

27.1 Introduction 48327.2 Role of Mass Spectrometry in Fungal Diagnostics 48427.3 The Importance of Secondary Fungal Metabolites: Mycotoxins and

Peptides 48627.4 Biological Activities of Cyclic Peptides 48927.5 Mass Spectrometry of Cyclic Peptides 49027.6 Concluding Remarks 496

28 Microarray Approaches to Autoantibody Profiling 511John M. Astle, Thomas Kodadek

28.1 Introduction 51128.2 Native Antigen Microarrays 51328.2.1 Protein Microarrays 51428.2.2 Peptide Microarrays 51628.2.3 Glycan Microarrays 51828.2.4 Lipid Microarrays 51828.2.5 Reverse-Phase Microarrays 51928.2.6 Antibody Microarrays 52128.3 Antigen Mimic Microarrays 52328.4 Antibody Microarrays in the Clinic 527

29 Identification of Tumor Antigen Directed Autoantibodies 533Sandra Faca, Sam Hanash

29.1 Introduction 53329.2 Proteomic Approaches for the Identification of Tumor Antigen

Directed Autoantibodies 53429.2.1 Some of the Potential Diagnostic Tumor Antigens Directed

Autoantibodies Identified by 2D western blot 53529.2.1.1 PGP 9.5 535

Contents XV

29.2.1.2 Annexins 1 and 2 53729.2.1.3 Calreticulin 53829.2.1.4 Other Potential Diagnostic Antigens Identified 53929.3 Development of a High-Throughput Microarray Approach

to Screen for Diagnostic Cancer Antigens 54029.3.1 Identification of UCH-L3 as a Potential Diagnostic Marker

for Colon Cancer Based on Screening Using Natural Protein

Microarray-Based Approach 54429.4 Conclusion 546

30 Antibody and Reverse Capture Protein Microarrays

for Clinical Proteomics 549Harvey B. Pollard, Ofer Eidelman, Meera Srivastava, Catherine Joswik,

Stephen Rothwell, Gregory P. Mueller, David M. Jacobowitz,

William B. Guggino, Jerry Wright, Pamela L. Zeitlin,

Cloud P. Paweletz

30.1 Introduction 54930.2 Antibody Microarrays 55030.2.1 A Technique in Development for Many Years 55030.2.2 Analysis of Patient Sera 55030.2.3 A Million Different Proteins in the Human Proteome 55230.2.4 Novel Expression Patterns for Hundreds of Known

Proteins 55230.3 Reverse Capture/Reversed-Phase Protein Microarrays 55330.3.1 A Massively Parallel Platform for Clinical Applications 55330.3.2 Discovery Platform for Signaling Pathway Analysis 55330.3.3 Peptide Arrays for Discovery of Autoantibodies 55530.4 Bioinformatics for Microarray Platforms 55630.4.1 Antibody Microarrays 55630.4.1.1 Overview 55630.4.1.2 Surfaces and Printers 55630.4.1.3 Detection Strategies 55730.4.1.4 Antibody Validation 55830.4.1.5 Normalization and Quantitation 55830.4.1.6 Interpretation 56330.4.2 Reverse Capture/Phase Protein Microarrays 56330.4.2.1 Overview 56330.4.2.2 Substratum Selection 56430.4.2.3 Detection Strategies 56430.4.2.4 Antibody Validation 56430.4.2.5 Normalization and Quantitation 56530.4.2.6 Interpretation 56530.5 Conclusions 566

XVI Contents

31 Use of Antibody Microarrays in the Analysis of Inflammation,

Autoimmunity, Viral Infection, and Cancer Metastases 571Rodney Lui, Angus Brown, Bosco Wu, Ming-Wei Lin, John Thompson,

Filip Braet, Wayne Dyer, JoDee Lattimore, Peter Macdonald,

Stephen Adelstein, Cristobal G. dos Remedios

31.1 Introduction 57131.1.1 Extracellular Proteins 57231.1.2 How the Antibody Array Works 57231.1.3 Numerical Analyses 57431.1.4 Comparison of Antibody Arrays with Flow Cytometry 57531.2 Inflammation 57531.2.1 Ischemic Heart Disease 57531.3 Autoimmune Disease 58231.3.1 Background to Systemic Lupus Eythematosus (SLE) 58231.3.2 Features of SLE 58231.4 Melanoma Cancer 58331.4.1 The Diagnostic Question 58331.4.2 Outlier Patients 58331.4.3 Hierarchical Clustering 58431.5 Vaccine Inflammation 58431.5.1 Smallpox Vaccination 58431.5.2 DotScan Data 58631.6 Concluding Remarks 588

VII Translation: Discovery to the Clinic 593

32 The Future: Translation from Discovery to the Clinic – Roles

of HUPO and Industry in Biomarker Discovery 595Gilbert S. Omenn, Peipei Ping

32.1 Introduction 59532.2 Brief Introduction of HUPO Initiatives 59632.3 General Lessons Illustrated with the Human Plasma Proteome

Project (HPPP) 59732.4 Brief Descriptions of Other HUPO Initiatives 60232.4.1 The Human Liver Proteome Project (HLPP) 60232.4.2 The HUPO Brain Proteome Project (HBPP) 60332.4.3 The HUPO Cardiovascular Initiative (HCVI) 60332.4.4 The Human Disease Glycomics/Proteome Initiative (HGPI) 60432.4.5 The HUPO Proteomic Standards Initiative (PSI) 60432.4.6 The HUPO Antibody Initiative (HAI) 60532.4.7 The Umbrella HUPO Biomarker Initiative has Several

Components 60532.4.7.1 The Human Kidney-Urine Proteome Project (HKUPP) 60532.4.7.2 The HUPO Cancer Biomarker Initiative 605

Contents XVII

32.4.7.3 HUPO Biomarkers Bioinformatics (BioBio) Initiative 60632.4.8 The HUPO/Invitrogen Test Sample Project 60632.5 General Strategies for the Use of Plasma or Serum for

Protein Biomarkers 60732.6 Pitfalls in Biomarker Discovery and Validation Studies 60832.7 Concluding Remarks 609

33 Requirements of a Good Biomarker: Translation

into the Clinical Laboratory 615Mario Plebani, Martina Zaninotto, Monica Maria Mion

33.1 Introduction 61533.2 Translational Research 61533.3 Biomarker: What Does It Mean? 61533.3.1 Biomarkers and Drug Development/Evaluation 61733.3.2 Biomarkers in Laboratory Medicine 61833.4 Development of Biomarkers: From Discovery to

Clinical Application 62133.5 Discovery 62233.5.1 Goal (Targeted)-Dependent Options 62333.5.2 Technologies 62333.6 Validation 62433.7 Standardization 62533.8 Clinical Association and Clinical Benefit 62633.9 Conclusions 628

34 Translation of Protein Biomarkers for Clinical Development 633Ian McCaffery, V. Dan Fitzpatrick, Shen Wu Wang, John M. Rossi,

Haifeng Bao, Sid V. Suggs, John Ferbas, Scott D. Patterson

34.1 Introduction 63334.2 Development of Biomarkers for Early Drug Development 63434.3 Marker of Biochemical Coverage – Development of an Assay of

Phospho S6 Protein as a PD Biomarker for Rapamycin Inhibition

of the mTOR Pathway 63834.3.1 PI3K/Akt/mTOR Pathway Background 63834.3.2 Therapeutics Directed at Inhibition of the mTOR Pathway 64034.3.3 Existing PBMC-Based PD Biomarker Assay of mTOR Inhibition 64034.3.4 Development of a Flow Cytometry Based PD Biomarker Assay and

Comparison of PBMC and Whole Blood Results 64134.3.5 Summary of Comparison of PD Biomarker Assays 64634.4 Discussion 64734.5 Summary 650

Index 653

XVIII Contents

Editor’s Overview

Clinical proteomic is an emerging discipline within proteomics that is exclusively

focused on its application to biomedicine. This book focuses on biomarker discovery

and validation and the pipeline required to increase the probability of success from the

view point of proteomics. It is hoped that sophisticated proteomic techniques and a

better understanding of basic pathophysiologies and protein chemistries will enhance

early disease detection, prognosis, risk stratification and therapeutic monitoring.

Clinical proteomics is a transdisciplinary field garnering aspects coming from

epidemiology, clinical chemistry, clinical medicine and proteomics. It is being built

on these disciplines, adapting experimental design, application, technologies and

knowledge to produce acceptable workflows. With respect to proteomics, success will

depend on the field developing and testing technologies and approaches for both

discovery and/or validation of biomarkers specifically in body fluids. The first section

of the book (Chapters 1–9) is composed of chapters dealing with the underlying

technologies which underpin proteomic applications – exploring the potential and

limitations of the various methods specifically for biomarker analysis. The subse-

quent sections highlight examples where proteomics is being applied in various

relevant clinical areas: cancer (Chapters 10–13); cardiovascular disease (Chapters 14–

19); vascular disease including stroke (Chapters 20–23); toxicity, bacterial and viral

assessment (Chapters 24–27); and autoantibody signatures (Chapters 28–31). In the

final section, three chapters deal with the broader context of clinical proteomics from

the perspective of clinical chemistry, academia and industry.

The dream of clinical proteomics is that patients’ lives are extended and the quality

of life can be improved by including proteomics in a clinicians’ diagnostic arsenal.

This book provides insight into the collective knowledge researchers have gained

over the last several years into a single resource book. It is aimed at addressing the

current issues surrounding the application of proteomics to the development of

biomarkers. There has been considerable hype around clinical proteomics and its

potential to change medicine. That potential remains. Nevertheless, there are many

challenges which need to be met.

Sincerely

The Editors

Michael Dunn and Jennifer Van Eyk

XIX

Short Summary

This is the first book to overview the emerging field of clinical proteomics with

sections focused on the latest technologies and summaries by leaders in the fields of

cancer, cardiovascular disease, toxicology, bacterial, viral and autoantibody assess-

ments. This book provides insight into the collective knowledge researchers have

gained over the last several years into a single resource book. It is aimed at

addressing the current issues surrounding the application of proteomics to the

development of biomarkers. There has been considerable excitement around clinical

proteomics and its potential to changemedicine. That potential remains but clinical,

technical and translational issues still need to be overcome.

Acknowledgements

The reasons for creating a book are numerous. The people to thanks are also

numerous. We thank the members of our labs who help to develop and create the

vision for biomarker discovery and our families, who provide the support to allow

those dream. Finally, we thank the authors who shared their insights and under-

standing.

XX Editor’s Overview

List of Contributors

Stephen Adelstein

Clinical Immunology

Royal Prince Alfred Hospital

Camperdown

Australia

Ruedi H. Aebersold

Institute of Molecular Systems

Biology,

ETH Zurich

and Faculty of Science

University of Zurich

Wolfgang-Pauli-Str. 16

8093 Zurich

Switzerland

N. Leigh Anderson

The Plasma Proteome Institute

Washington DC 20009-3450

USA

John M. Arthur

Medical University of

South Carolina

Division of Nephrology

PO Box 250623

Charleston, SC 29425-2220

USA

John M. Astle

Departments of Internal Medicine

and Molecular Biology

University of Texas Southwestern

Medical Center

5323 Harry Hines Blvd

Dallas, Texas 75390-9185

USA

Brian M. Bagley

Calibrant Biosystems,

910 Clopper Road,

Suite 220N,

Gaithersburg

MD 20878

USA

Rosamonde E. Banks

Cancer Research UK

Clinical Centre

St James’s University Hospital

Beckett Street

Leeds LS9 7TF

UK

Haifeng Bao

Amgen, Inc.

Molecular Sciences

One Amgen Center Drive,

Mail Stop 1-1A

Thousand Oaks, CA 91320-1799

USA

XXI

Timothy J. Barder

Eprogen, Inc.,

8205 S. Cass Ave. Ste. 106,

Darien, IL 60561

USA

Luca Bini

Department of Molecular Biology

University of Siena

Via Fiorentina

153100 Siena

Italy

Mary E. Bollard

Department of Biochemistry

University of Cambridge

Tennis Court Road

Cambridge

UK

Filip Braet

Australian Key Centre for

Microscopy and Microanalysis

The University of Sydney

Sydney 2006

Australia

Teresa A. Brentnall

Department of Medicine

University of Washington

1959 NE Pacific St

Seattle, WA 98195

USA

Angus Brown

Bosch Institute (F13)

The University of Sydney

Sydney 2006

Australia

D. Allan Butterfield

Center of Membrane Sciences

University of Kentucky

Lexington, KY 40506

USA

Valerie S. Calvert (12)

Center for Applied Proteomics and

Molecular Medicine

George Mason University

10900 University Blvd, MS 4E3

Manassas, VA 20110

USA

Richard M. Caprioli

Department of Biochemistry

Vanderbilt-Ingram Comprehensive

Cancer Center

9160 MRB III

Vanderbilt University

School of Medicine

Nashville, TN 37232-8575

USA

Julio E. Celis

Inst. Cancer Bio/Human

Genome Res

Danish Cancer Society

Strandboulevarden 49

2100 Copenhagen

Danmark

Daniel W. Chan

Biomarker Discovery Center

Johns Hopkins University

Baltimore, MD

USA

Ru Chen

Department of Medicine

University of Washington

1959 NE Pacific St

Seattle, WA 98195

USA

Songming Chen

The Van Andel Research Institute

333 Bostwick

Grand Rapids, MI 49503

USA

XXII List of Contributors

David E Clemmer

Department of Chemistry

Indiana University

800 E. Kirkwood Ave.

Bloomington, IN 47405

USA.

Garry L. Corthals

Biomedical Proteomics Research

Group

Geneva University Hospital/LCCC

24 rue Micheli-du-Crest

1211 Genève 14

Switzerland

David R. Cotter

Johns Hopkins University

School of Medicine

725 N. Wolfe St.

B7 Biophysics

Baltimore, MD 21205

USA

Robert J. Cotter

Johns Hopkins University

School of Medicine

725 N. Wolfe St.

B7 Biophysics

Baltimore, MD 21205

USA

Jacques Deshusses

Biomedical Proteomics Research

Group,

Department of Structural Biology

and Bioinformatics,

Geneva University,

1 Michel Servet,

1211 Geneva,

Switzerland

Don L. DeVoe

Department of Mechanical

Engineering and Bioengineering

Program

University of Maryland,

College Park, MD 20742

USA

Pamela M. Donoghue

Immune Regulation Research

Group

Trinity College

Dublin 2

Ireland

Cristobal G. dos Remedios

Bosch Institute (F13)

The University of Sydney

Sydney 2006

Australia

Michael J. Dunn

University College Dublin

Proteome Research Centre

UCD Conway Institute of

Biomolecular and Biomedical

Research

Dublin 4

Ireland

Wayne Dyer

Australian Red Cross Blood Service

Clarence Street

Sydney 2000

Australia

Marta Dziechciarkova

Palacky University and

University Hospital Olomouc

Faculty of Medicine and Dentistry

Puskinova 6

77520 Olomouc

Czech Republic

List of Contributors XXIII

Petr Dzubak

Faculty of Medicine and Dentistry

Palacky University and University

Hospital Olomouc

Puskinova 6

77520 Olomouc

Czech Republic

Kirsten H. Edmiston

Center for Applied Proteomics and

Molecular Medicine

George Mason University

10900 University Blvd, MS 4E3

Manassas, VA 20110

USA

Ofer Eidelman

Department of Anatomy,

Physiology and Genetics

USU School of Medicine (USUHS)

4301 Jones Bridge Road

Bethesda, MD 20814

USA

Andrew Emili

Banting and Besst Department of

Medical Research and Department

of Medical Genetics and

Microbiology

Donnelly Centre for Cellular and

Biomolecular Research

University of Toronto

Canada

Jane English

Royal College of Surgeons in

Ireland

Department of Psychiatry

Education and Research Centre

Beaumont Hospital

Dublin 9

Ireland

Virginia Espina

Center for Applied Proteomics and

Molecular Medicine

George Mason University

10900 University Blvd, MS 4E3

Manassas, VA 20110

USA

Sandra Faca

Fred Hutchinson Cancer Research

Center

1100 Fairview Avenue N., M5-C800

P.O. Box 19024

Seattle, WA 98109

USA

Xueping Fang

Department of Chemistry and

Biochemistry

University of Maryland,

College Park, MD 20742

USA

John Ferbas

Amgen, Inc.

Clinical Immunology

One Amgen Center Drive,Mail Stop

30E-3-C

Thousand Oaks, CA 91320-1799

USA

Roisean E. Ferguson

Cancer Research UK Clinical

Centre

St James’s University Hospital

Beckett Street

Leeds LS9 7TF

UK

XXIV List of Contributors

V. Dan Fitzpatrick

Amgen, Inc.

Molecular Sciences

One Amgen Center Drive, Mail

Stop 1-1A

Thousand Oaks, CA 91320-1799

USA

Melanie Föcking

Royal College of Surgeons in

Ireland

Department of Psychiatry

Education and Research Centre

Beaumont Hospital

Dublin 9

Ireland

Qin Fu

Department of Medicine

Johns Hopkins Bayview Proteomics

Center

Johns Hopkins University

Baltimore, MD 21224

USA

Suresh Jivan Gadher

Beckman Coulter International S.A.

22, rue Juste Olivier, Casa Postale

1059

1260 Nyon 1

Switzerland

Robert E. Gerszten, M.D.

Cardiology Division and Center for

Immunology and Inflammatory

Diseases

Massachusetts General Hospital

East-8307

149 13th Street

Charlestown, MA 02129

USA

Anastassios Giannakopulos

Institute of Microbiology

Academy of Sciences of the

Czech Republic

Videnska 1083

142 20 Prague

Czech Republic

David R. Graham

Bayview NHLBI Proteomics Center

Johns Hopkins School of Medicine

Bayview Campus

5200 Eastern Avenue

Baltimore, MD 21224

USA

Anthony Gramolini

Department of Physiology and

Heart and Stroke/Richard Lewar

Center of Excellence

University of Toronto

112 College St, Rm 307

Toronto, Ontario

Canada

William B. Guggino

Department of Physiology,

Johns Hopkins University

School of Medicine,

JHMI,

Baltimore, MD 21205

USA

Rebekah L. Gundry (18)

Johns Hopkins University

School of Medicine

725 N. Wolfe St.

B7 Biophysics

Baltimore, MD 21205

USA

Brian B. Haab

The Van Andel Research Institute

333 Bostwick

Grand Rapids, MI 49503

USA

List of Contributors XXV

Marian Hajduch

Faculty of Medicine and Dentistry

Palacky University and University

Hospital Olomouc

Puskinova 6

77520 Olomouc

Czech Republic

Petr Halada

Institute of Microbiology

Czech Academy of Sciences

Videnska 1083

14220 Praha

Czech Republic

Samir M. Hanash

Fred Hutchinson Cancer Research

Center

1100 Fairview Avenue N., M5-C800

P.O. Box 19024

Seattle, WA 98109

USA

Gerald Hart

725 N Wolfe Street

Biochemistry Department

Baltimore, MD 21205

USA

Vladimir Havlicek

Institute of Microbiology

Academy of Sciences of the Czech

Republic

Videnska 1083

142 20 Prague

Czech Republic

Jan Hirsh

Department of Anesthesia and

Perioperative Medicine

University of California

505 Parnassus Ave. HSW 825

San Francisco, CA 94143-0130

USA

Denis F. Hochstrasser

Biomedical Proteomics Research

Group

Clinical Chemistry Laboratory

Geneva University Hospital

Geneva

Switzerland

Don Hunt

Chemistry Department

University of Virginia

Charlottesville, VA 22908

USA

David M. Jacobowitz

Laboratory of Clinical Science,

National Institute for Mental

Health,

Bethesda, MD 20892

USA

Jon M. Jacobs

Biological Sciences Division

Pacific Northwest National

Laboratory

P. O. Box 999

Richland, WA 99352

USA

Alexandr Jegorov

Institute of Microbiology

Academy of Sciences of the Czech

Republic

Videnska 1083

142 20 Prague

Czech Republic

XXVI List of Contributors

Robert L. Jesse

Department of Internal Medicine

Cardiology Division

Medical College of Virginia

Virginia Commonwealth University

Veterans Health Administration

12th and Marshall Streets

Richmond, VA 23298-0051

USA

Catherine Joswik

Department of Anatomy

Physiology and Genetics

USU School of Medicine (USUHS)

4301 Jones Bridge Road

Bethesda, MD 20814

USA

Urban A. Kiernan

Intrinsic Bioprobes, Inc.

2155 E. Conference Drive, Suite 104

Tempe, AZ 85284

USA

Thomas Kodadek

University of Texas

Southwestern Medical Center

Departments of Internal Medicine

and Molecular Biology

5323 Harry Hines Blvd

Dallas, TX 75390-9185

USA

Hana Kovarova

Czech Academy of Sciences

Institute of Animal Physiology and

Genetics

Rumburska 89

27721 Libechov

Czech Republic

JoDee Lattimore

Sydney Melanoma Unit

Royal Prince Alfred Hospital

Camperdown

Australia

Cheng S. Lee

Department of Chemistry and

Biochemistry

University of Maryland

College Park, MD 20742

USA

Gregory D. Lewis

Center for Immunology and

Inflammatory Diseases

Massachusetts General Hospital

Charlestown, MA and Harvard

Medical School

Boston, MA

USA

Daniel C. Liebler

Southwest Environmental Health

Sciences Center

College of Pharmacy

University of Arizona

Tucson, AZ 85721-0207

USA

Ming-Wei Lin

Clinical Immunology

Royal Prince Alfred Hospital

Camperdown

Australia

Lance A. Liotta

Center for Applied Proteomics and

Molecular Medicine

George Mason University

10900 University Blvd, MS 4E3

Manassas, VA 20110

USA

David M. Lubman

Department of Surgery

1150 West Medical Center Drive

Ann Arbor, MI 48109-1055

USA

List of Contributors XXVII

Rodney Lui

Bosch Institute (F13)

The University of Sydney

Sydney 2006

Australia

Peter MacDonald

Cardiopulmonary Transplant Unit

St. Vincent’s Hospital

Darlinghurst, 2010

Australia

Ian McCaffery

Amgen, Inc.

Molecular Sciences

One Amgen Center Drive,

Mail Stop 1-1A

Thousand Oaks, CA 91320-1799

USA

Dr. Ciara A. McManus

University College Dublin

Proteome Research Centre

UCD Conway Institute of

Biomolecular and Biomedical

Research

Dublin 4

Ireland

Michael A. Matthay

Department of Anesthesia and

Perioperative Medicine

University of California

505 Parnassus Ave. HSW 825

San Francisco, CA 94143-0130

USA

Bruce Alex Merrick

Proteomics Group

National Institute of Environmental

Health Sciences

P.O. Box 12233

Research Triangle Park, NC 27709

USA

Monica Maria Mion

Department of Laboratory Medicine

University-Hospital

Via Giustiniani 2

35128 Padova

Italy

Harald Mischak

Mosaiques-Diagnostics and

Therapeutics AG

Mellendorfer Str. 7-9

30173 Hannover

Germany

David E. Misek

University of Michigan

Department of Surgery

1150 West Medical Center Drive

Ann Arbor, MI 48109-1055

USA

Gregory P. Mueller

Department of Anatomy,

Physiology and Genetics

USU School of Medicine (USUHS)

4301 Jones Bridge Road

Bethesda, MD 20814

USA

Dobrin Nedelkov

Intrinsic Bioprobes, Inc.

2155 E. Conference Drive, Suite 104

Tempe, AZ 85284

USA

Jan Nedvěd

Institute of Microbiology

Academy of Sciences of the Czech

Republic

Videnska 1083

142 20 Prague

Czech Republic

XXVIII List of Contributors