Methods in Animal Proteomics E-Book

Philip Whitfield

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 883

Veröffentlichungsjahr: 2011

Beschreibung

The study of proteomics provides researchers with a better understanding of disease and physiological processes in animals. Methods in Animal Proteomics will provide animal scientists and veterinarians currently researching these topics in domestic animals a firm foundation in the basics of proteomics methodology, while also reviewing important advances that will be of interest to established researchers in the field. Chapters will provide practical information on a range of topics including protein identification and separation, bioinformatics, and applications to disease and reproduction research. This text will be written by leading international proteomics experts and essential for researchers in the fields of animal biology and veterinary medicine.

Leseprobe

Table of Contents

Cover

Title page

Copyright page

Contributors

Acknowledgments

Section 1: Exploring Animal Proteomes

1  An Introduction to Animal Proteomics

1.1 Proteomics and Animal Systems

1.2 Exploring Animal Proteomes

1.3 Applications of Proteomics in Animal Systems

2  Types of Sample and Experimental Planning

2.1 Introduction

2.2 Types of Samples and Their Collection

2.3 General Preparation of Body Fluids, Cultured Cells in Suspension, and Blood

2.4 Homogenization, Clarification, and Simplification

2.5 Determination of Protein Concentration in Proteomic Samples

2.6 Enzymatic Digestion

2.7 Experimental Design: Strategies for Simplification and Enrichment

2.8 Specialized Techniques for Sample Preparation

2.9 Experimental Planning and Design: General Guidelines

2.10 Biomarker Discovery: Special Considerations for Experimental Planning

2.11 Concluding Remarks

3  Protein Separation Strategies

3.1 Introduction

3.2 “Classical” 2DE

3.3 Other Varieties of 2DE

3.4 Sample Complexity

3.5 The Influence of Staining

3.6 “Post-detection”

3.7 Future Trends

4  Methods and Approaches to Mass Spectroscopy-Based Protein Identification

4.1 Introduction

4.2 MS

4.3 Sample Preparation

4.4 Protein Identification

4.5 Linking Mass Spectra with Proteins

4.6 Validation of MS Data

4.7 Conclusions

5  Bioinformatics in Animal Proteomics

5.1 Introduction

5.2 Methods

5.3 Future Trends

5.4 Sources of Further Information

6  Comparative Proteomic Approaches

6.1 Global Study of Proteins

6.2 Approaches to Characterize the Proteome

6.3 Quantitative Proteomics

6.4 Bioinformatics and Biostatistics

6.5 Future Prospects

7  Advancing Technologies for Spatial and Temporal Proteomics

7.1 Introduction

7.2 Investigating the Spatial Proteome

7.3 Temporal Proteomics

7.4 Case Study: Spatial and Temporal Profiling in the Chicken

7.5 Combining Spatial and Temporal Proteomics

7.6 Recent Developments in Animal Proteomics

7.7 Future Trends

7.8 Conclusions

7.9 Sources of Further Information

Section 2: Applications of Proteomics in Animal Biology

8  Proteomic Strategies to Investigate Adaptive Processes

8.1 Introduction

8.2 Hibernation Physiology and Behavior

8.3 Sampling Strategy

8.4 Sample Collection and Storage

8.5 Design and Execution of a Quantitative Proteomic Experiment

8.6 Conclusion

9  Investigation of Animal Venoms and Toxins

9.1 Introduction

9.2 Natural Bioresources

9.3 Pharmaceutical Success Stories

9.4 The Methods in Toxin Sequencing

9.5 Reducing Complexity by Chromatography

9.6 Bioassay Considerations

9.7 Classical Edman

9.8 Overview of MS Methods for Chemical Prospecting

9.9 Soft Ionization Methods

9.10 Common Mass Analyzers for Peptide Analysis

9.11 Information Obtained from Different MS Systems

9.12 Cloning Studies

9.13 New Developments and Future Trends

9.14 Conclusions

9.15 Other Sources of Information

10  Proteomics in Animal Health and Disease

10.1 Introduction

10.2 Proteomics of Plasma and Serum of Animals

10.3 Proteomics of the Liver of Animals

10.4 Proteomics of the Immune System of Animals

10.5 Proteomics of the CNS in Animals

10.6 Proteomics of Muscle in Animals

10.7 Proteomics of Milk and the Mammary Gland of Animals

10.8 Proteomics of the Intestine in Animals

10.9 Proteomics of Skin in Animals

10.10 Proteomics of the Respiratory Organs in Animals

10.11 Proteomics of the Visual Apparatus in Animals

10.12 Proteomics of Bone and Cartilage in Animals

10.13 Proteomics of Endocrinology in Animals

10.14 Proteomics of Kidney and Urine in Animals

10.15 Conclusions

11  Application of Proteomics to Elucidate Bacterium–Host Interactions

11.1 Characterizing Bacterial Proteomes

11.2 Employing Surrogate Conditions

11.3 Immunoproteomics

11.4 In Vitro Challenge Systems

11.5 Pathogen–Host Interaction In Vivo

11.6 Concluding Comments

12  Animal Parasitology and Proteomics

12.1 Introduction

12.2 Proteomic Technologies

12.3 Parasite Proteomics

12.4 The Potential of Comparative Proteomic Analysis

12.5 Understanding Drug Action and Resistance

12.6 Downstream of the Proteome: Metabolomics

12.7 Global Approaches and the Search for Biomarkers

12.8 Future Trends

13  Proteomics in Animal Reproduction and Breeding

13.1 Introduction

13.2 Proteomics

13.3 Proteomics and Female Fertility

13.4 Proteomics and Male Fertility

13.5 Sperm–Oocyte Plasma Membrane Binding and Fusion

13.6 Biomarkers

13.7 Conclusions

14  Assessment in the Quality and Safety of Food of Animal Origin

14.1 Introduction

14.2 Proteomics to Assess the Quality of Muscle Foods

14.3 Proteomics to Assess the Quality of Dairy Products

14.4 Proteomics and Species Authentication

14.5 Identification and Characterization of Allergens of Animal Origin

14.6 Conclusions

Acknowledgments

Index

This edition first published 2011 © 2011 by John Wiley & Sons, Inc.

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Library of Congress Cataloging-in-Publication Data

Methods in animal proteomics / editors, P. David Eckersall, Phillip D. Whitfield.

p. cm.

 Includes bibliographical references and index.

 ISBN-13: 978-0-8138-1791-0 (hardcover : alk. paper)

 ISBN-10: 0-8138-1791-9

 1. Proteomics–Methodology. 2. Veterinary medicine–Methodology. I. Eckersall, P. D. II. Whitfield, Phillip D.

QP551.M3863 2011

636.089'2398–dc22

2011012035

A catalogue record for this book is available from the British Library.

This book is published in the following electronic formats: ePDF 9780470960639; Wiley Online Library 9780470960660; ePub 9780470960646; Mobi 9780470960653

Contributors

Paul BlakeleyUniversity of ManchesterUKRichard BurchmoreUniversity of GlasgowUKShane C BurgessMississippi State UniversityMS, USAAlessio Di LuccaTeagasc, Ashtown Food Research CentreIrelandMary K. DohertyUniversity of the Highlands and IslandsUKP. David EckersallUniversity of GlasgowUKL. Elaine EppersonUniversity of Colorado School of MedicineCO, USASimon J. HubbardUniversity of ManchesterUKAndrew R. JonesUniversity of LiverpoolUKKathryn S. LilleyUniversity of CambridgeUKJohn D. LippolisNational Animal Disease CenterIA, USAJingshun LiuTeagasc, Ashtown Food Research CentreIrelandBegonya MarcosTeagasc, Ashtown Food Research CentreIrelandSandy L. MartinUniversity of Colorado School of MedicineCO, USAMark McLaughlinUniversity of GlasgowUKStephen McCLeanUniversity of UlsterUKErdogan MemiliMississippi State UniversityMS, USAIngrid MillerUniversity of Veterinary MedicineVienna, AustriaAnne Maria MullenTeagasc, Ashtown Food Research CentreIrelandChristine OlverColorado State UniversityCO, USADivyaswetha PeddintiMississippi State UniversityMS, USADilip K. RaiTeagasc, Ashtown Food Research CentreIrelandJohanna S. ReesUniversity of CambridgeUKTimothy A. ReinhardtNational Animal Disease CenterIA, USADavid G.E. SmithMoredun Research InstituteUKPhillip D. WhitfieldUniversity of the Highlands and IslandsUKJames C. WrightWellcome Trust Sanger InstituteUK

Acknowledgments

PDW would like to thank former colleagues at the University of Liverpool and his current group at the University of the Highlands and Islands. The discussions with Rob Beynon have been invaluable. In addition, PDW thanks Mary Doherty for her considerable help and assistance in putting the book together. The financial support from Biotechnology and Biological Sciences Research Council (BBSRC) is also gratefully acknowledged.

We would like to convey our gratitude to the editorial team of Shelby Allen, Susan Engelken, Anna Ehler, and Justin Jeffryes at Wiley-Blackwell for all their advice and support. Finally, we would like express our great thanks to all the authors who have contributed to the book. Without their expertise and commitment this project could never have been realized.

P. David Eckersall

Phillip D. Whitfield

Section 1: Exploring Animal Proteomes

1

An Introduction to Animal Proteomics

Phillip D. Whitfield and P. David Eckersall

1.1 Proteomics and Animal Systems

Proteomics is conventionally described as the study of the protein component of a cell, a tissue, or an organism at a given time under given conditions (Wilkins et al., 1996). It complements and extends the study of genomes and transcript data, reflecting the true biochemical outcome of genetic information. However, proteomics has developed from cataloguing proteins to an advanced discipline that requires a substantial investigation of the protein world, defining the quantities, posttranslational variants, binding partners, and intracellular stability of proteins in biological systems (Doherty and Beynon, 2006).

The exquisite sensitivity and selectivity of contemporary protein analysis means that proteomics is at the forefront of biological and biomedical research. Perhaps not surprisingly, investigations have often been focused on prevalent and important human diseases such as cardiovascular disease, neurological disorders, and cancer. In comparison, proteomic investigations aimed at enhancing our knowledge of animal biology have had a much lower profile. In this book, we have brought together a group of researchers in an attempt to provide an overview of the opportunities and challenges within the emerging field of animal proteomics. It is by no means exhaustive but is aimed at capturing the excitement of current practitioners of the field and relates to their experiences. The book addresses the experimental strategies and techniques employed in animal proteomics studies. It also outlines key applications of proteomics to the study of animal systems across a variety of disciplines. Importantly, the focus of the book extends beyond the use of laboratory rodent models and instead encompasses a broad range of companion and production animals, birds, fish, reptiles, and wildlife.

1.2 Exploring Animal Proteomes

Proteomics has the ability to encompass the large-scale identification, characterization, and quantification of the proteins in animal systems. The advances made in proteomics have been underpinned by significant technical developments, which have revolutionized protein analysis. The investigation of animal proteomes requires a combination of efficient and stringent separation technologies, high-resolution mass spectrometry, and powerful bioinformatic tools to characterize a broad range of proteins (Lopez, 2007; Han et al., 2008; Kumar and Mann, 2009). However, the use of proteomic strategies in animals brings significant practical and analytical challenges.

There is a wide range of biofluids and tissue samples that can be been employed in animal proteomic experiments. These include plasma, serum, blood cells, urine, cerebrospinal fluid, amniotic fluid, synovial fluid, seminal fluids, bile, feces, saliva, milk, eggs, wool, and venom as well as many different tissue types, which reflect the diverse anatomy and physiology of animal species. The tissues under investigation might have components that are normally not found in more commonly studied specimens (e.g., lipids in milk), affecting the quality of the analysis and the reproducibility of the results. An additional issue is the enormous complexity and extensive dynamic range of protein concentrations in the body fluids and tissues of animals. There is often an overrepresentation of a few proteins; for example, in serum there are several orders of magnitude difference in the concentration range of the highest and lowest abundant proteins, while in tissues this range is usually smaller. To reduce the complexity and allow study of proteins of lower abundance, different depletion strategies or prefractionation methods are required. Christine Olver in Chapter 2 discusses the key considerations that have to be given to the experimental design, the preparation and extraction of proteins from different sample types, as well as the most appropriate methodologies to be used in the analysis of the protein complement.

A variety of powerful experimental approaches exist for profiling of animal proteomes. In Chapter 3 Ingrid Miller provides a comprehensive review of the principles and technical aspects relating to electrophoretic and chromatographic techniques that are routinely used for protein separation and isolation. The chapter also outlines how these tools may be employed to discover proteins that are differentially expressed in animal systems. In proteomic experiments proteins are typically identified using mass spectrometry as discussed by Lippolis and Reinhardt (Chapter 4). The most common strategy involves the analysis of peptides rather than intact proteins. In this process, proteins of interest, either in-solution or excised from a gel, are digested with a proteolytic enzyme, typically trypsin, and the resultant peptides are analyzed by mass spectrometry. A process referred to as peptide mass fingerprinting (PMF) utilizes the capability of matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to produce a unique pattern of peptide ions for individual proteins. These proteins are then identified by matching the list of experimental peptide ion masses with the theoretical calculated peptide masses obtained from in silico digestion of all proteins in a given database.

A potential difficulty with this approach is the lack of complete and annotated genome sequences, which can result in an under-representation in protein sequence databases of many animal species. While it is possible to identify proteins with high sequence conservation via cross-species matching (Wright et al., 2010), amino acid changes in a protein can result in a different PMF. To accurately determine the identity of proteins from animals often requires de novo sequencing of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In LC-MS/MS experiments sequences of peptides can be matched to comprehensive protein databases using different search algorithms. This approach has been successfully used to identify proteins in animals where little or no sequence information exists, although it still relies on sufficient sequence information being available from a homologous protein in another sequenced species. However, the publication of the genome sequences of important animal species, such as the chicken (Hillier et al., 2004), dog (Lindblad-Toh et al., 2005), cow (Elsik et al., 2009), and horse, (Wade et al., 2009) and their annotation will facilitate the enhanced interpretation of proteomic experiments, minimizing the requirement for cross-species matching and de novo sequencing. This should improve confidence in the protein identifications provided by a typical proteomic experiment and provide the basis for further exploration of animal proteomes.

“Shotgun proteomics” has also emerged as a powerful technique for the analysis of complex protein mixtures pioneered by methods such as multidimensional protein identification technology (MudPIT) (Washburn et al., 2001). This methodology analyzes protein-derived peptides that are subjected to strong-cation exchange (SCX) chromatography, and online reverse-phase separation prior to mass spectrometric analysis. Alternative shotgun approaches, which involve one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1-D SDS-PAGE) coupled with LC-MS/MS have also been developed as part of strategies aimed at the routine qualitative identification of proteins (Schirle et al., 2003).

While the high-throughput nature of shotgun proteomics approaches has gained significant popularity, it should be noted that protein identification by this method is still very challenging. From a single experiment large amounts of data are generated, which must be assembled to give protein identification. As a result, robust bioinformatic and data-handling methods are required to extract the maximal amount of meaningful information (Kislinger and Emili, 2005). Large-scale proteomics using LC-MS/MS and automated database searching is prone to an increase in the number of incorrect peptide identifications. In addition, insufficient protein sequence coverage and sequence redundancy, that is, the same peptide sequence can be present in multiple different proteins, often preclude discrimination between protein isoforms or closely related proteins in the absence of information about the mature forms. Further, gross or subtle changes in the protein/peptide sequence can lead to proteins remaining unidentified, which has significant implications for biological interpretation of proteomic data.

Novel mass proteomic approaches are also now emerging to enhance protein identification by characterizing specific regions of proteins. Positional proteomic methods aim to simplify the proteome by isolating either the C- or N-terminal peptides (Gevaert et al., 2003; Nakazawa et al., 2008) and subjecting these peptides to LC-MS/MS analysis. As the position of each peptide is known in the protein, it is possible to minimize the bioinformatic search space, facilitating a more confident protein assignment from a single peptide. Many of these bioinformatic issues associated with identification of proteins from animal body fluids and tissues are discussed by Blakeley and colleagues in Chapter 5.

Experimental approaches to compare protein profiles between animal samples and characterize those proteins that exhibit differential expression are discussed by Rees and Lilley (Chapter 6). Densitometric image analysis of gels, where protein densities/volumes determine the relative changes in protein expression between differing states (Unlu et al., 1997), has been extensively used. Increasingly, mass spectrometric-based approaches in which the amounts of protein are defined either relative to a comparator system or in absolute terms (Elliott et al., 2009; Pan et al., 2009) are now being employed. The area of quantitative proteomics has been further extended through the study of proteome dynamics. In Chapter 7, Mary Doherty outlines the development of proteomic strategies to probe the spatial and temporal proteome. This includes novel methods to define the turnover of individual proteins in animal systems. The potential of these approaches to provide additional insights into the mechanism of change between physiological states is also discussed.

1.3 Applications of Proteomics in Animal Systems

The application of proteomic technologies to the study of animal systems has relevance to researchers in a number of fields including basic and clinical animal sciences, food science, and agriculture.

Animals rarely exist in unchanging environments and many external factors can dominate their life strategies. A perspective driven by proteomics can provide an integrated approach that encompasses a global view of protein expression in animal tissues under different environmental conditions. In Chapter 8 Epperson and Martin outline studies that have employed proteomic technologies to explore the molecular basis of adaptive processes in animals. Similarly, some animal species have also evolved unique defense mechanisms. Animal venoms and toxins contain complex mixtures of proteins and peptides. Stephen McClean in Chapter 9 details the way in which proteomic technologies are now being used to characterize the active components of venoms and toxins from animals and investigate their biological and pharmacological activities (Escoubas and King, 2009).

The ability to obtain a profile of the biochemical responses at the protein level may have direct outcomes in improving our understanding of animal health and disease (Moore et al., 2007). From a veterinary perspective the optimization of animal health is clearly a motivating factor. As discussed by Eckersall and McLaughlin (Chapter 10), the advancement of proteomic technologies has added new dimensions to the analyses of clinically relevant samples from animals and these strategies are increasingly being used to identify diagnostic biomarkers and investigate the etiology of animal disease states.

Animals are constantly under challenge by pathogens such as bacteria and parasites. In particular, infectious diseases can adversely impact on the management of livestock, poultry, and fish, resulting in huge production losses, which is of major importance to agriculture. Pathogens are likely to have profound effects on the cells that they invade and may be reflected in an altered expression of a broad range of proteins at the cellular, tissue, and system levels. In their chapters, Smith (Chapter 11) and Burchmore (Chapter 12) outline the way in which proteomic approaches are being used to determine the host’s response to infection, investigate the mechanisms of transmission of infectious diseases, and develop novel strategies for therapeutic intervention including vaccine candidates. Proteomic technologies are also being utilized to study animal fertility and reproduction. Peddinti and colleagues (Chapter 13) detail the use of proteomics to expand our understanding of the oocyte, spermatozoon, and embryo in animal species and how this information may enhance breeding programs. In addition to live animals, the way in which products of animal origin such as meats, milk, and cheese are produced and processed is a major consideration (Pischetsrieder and Baeuerlein, 2009). In Chapter 14 Marcos and colleagues discuss the use of proteomic strategies to monitor food composition, authenticity, and safety and provide a means to define meat and fish quality, detect food allergens, and identify markers of spoilage in dairy products.

We are very grateful to of the all the authors who have readily contributed their expertise and insights to this volume. The book aims to act as an introductory text for animal scientists with little or no experience of proteomics, while providing an up-to-date reference for researchers with a background in the area. We hope that readers will find the book interesting and that it proves to be a useful source of information for anyone working in the growing field of animal proteomics.

References

Doherty, M.K. and Beynon, R.J. 2006. Protein turnover on the scale of the proteome. Expert Rev. Proteomics 3(1):97–110.

Elliott, M.H., et al. 2009. Current trends in quantitative proteomics. J. Mass Spectrom. 44(12):1637–1660.

Elsik, C.G., et al. 2009. The genome sequence of taurine cattle: a window to ruminant biology and evolution. Science 324(5926):522–528.

Escoubas, P. and King, G.F. 2009. Venomics as a drug discovery platform. Expert Rev. Proteomics 6(3):221–224.

Gevaert, K., et al. 2003. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nat. Biotechnol. 21(5):566–569.

Han, X., et al. 2008. Mass spectrometry for proteomics. Curr. Opin. Chem. Biol. 12(5):483–490.

Hillier, L.W., et al. 2004. Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432(7018):695–716.

Kislinger, T. and Emili, A. 2005. Multidimensional protein identification technology: current status and future prospects. Expert Rev. Proteomics 2(1):27–39.

Kumar, C. and Mann, M. 2009. Bioinformatics analysis of mass spectrometry-based proteomics data sets. FEBS Lett. 583(11):1703–1712.

Lindblad-Toh, K., et al. 2005. Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature 438(7069):803–819.

Lopez, J.L. 2007. Two-dimensional electrophoresis in proteome expression analysis. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 849(1-2):190–202.

Moore, R.E., et al. 2007. Biomarker discovery in animal health and disease: the application of post-genomic technologies. Biomark. Insights 2:185–196.

Nakazawa, T., et al. 2008. Terminal proteomics: N- and C-terminal analyses for high-fidelity identification of proteins using MS. Proteomics 8(4):673–685.

Pan, S., et al. 2009. Mass spectrometry based targeted protein quantification: methods and applications. J. Proteome Res. 8(2):787–797.

Pischetsrieder, M. and Baeuerlein, R. 2009. Proteome research in food science. Chem. Soc. Rev. 38(9):2600–2608.

Schirle, M., et al. 2003. Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry. Mol. Cell. Proteomics 2(12):1297–1305.

Unlu, M., et al. 1997. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18(11):2071–2077.

Wade, C.M., et al. 2009. Genome sequence, comparative analysis, and population genetics of the domestic horse. Science 326(5954):865–867.

Washburn, M.P., et al. 2001. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19(3):242–247.

Wilkins, M.R., et al. 1996. From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Biotechnology (NY) 14(1):61–65.

Wright, J.C., et al. 2010. Cross species proteomics. Methods Mol. Biol. 604:123–135.

2

Types of Sample and Experimental Planning

Christine Olver

2.1 Introduction

The proteome is the full complement of proteins expressed by a cell or tissue at any given time and/or any given environmental condition (Aebersold and Mann, 2003; Cristea et al., 2004). Proteomics is the study and/or identification of this set or a subset of these proteins. For biological samples, including tissues, cells, and fluids, the proteins must first be extracted from cells and solubilized in appropriate media. This process includes disrupting tissues and cells (lysis), homogenizing the crude lysate, solubilizing the proteins, simplification or fractionation, and removing interfering substances.

Experiments must be planned carefully with goals and analytical methods in mind so that the appropriate tissues, cells, cell compartments, solubilization method, and digestion enzyme can be chosen. Because of the complexity and large dynamic range of protein concentrations in tissues, samples are quite frequently fractionated prior to analysis. This initial fractionation may be a gel-based targeted approach, or a liquid-based nontargeted (“shotgun”) approach. The sample preparation protocol must be tailored to this initial separation process. For instance, if your experiment will involve an initial separation using two-dimensional polyacrylamide gel electrophoresis (2D PAGE), it is important that the sample is prepared without charged molecules such as salts or ionic detergents, or that a “cleanup” procedure is performed to remove such substances. If the goal of the experiment is to identify membrane proteins, the sample preparation protocol should enrich for those proteins. This chapter provides a broad overview of sample types, sample collection and processing, cell lysis and solubilization, and preparation for mass spectrometry, and briefly mentions some specialized techniques for protein targeting and/or enrichment. It is always wise to research previously described methods for your sample type and experimental plan. The reader is also directed to additional reference materials at the end of this chapter.

2.2 Types of Samples and Their Collection

Samples for proteomic experiments include whole tissues, cells in culture medium, body fluids containing soluble proteins as well as particulate matter (cells or cell vesicles), and cell-free fluids (serum or plasma). Figure 2.1 shows a cartoon of various preparation strategies for the different types of samples.

Fig. 2.1 The general scheme of sample preparation for proteomics experiments. Biological samples must be prepared crudely, and then undergo a series of refining and processing steps, as described in the text.

2.2.1 Whole Tissue

Examples of whole tissue include tumor biopsies, whole organs, and sections of whole organs. Regardless, whole tissue should be processed quickly after harvest, and in cold conditions, to avoid secondary changes such as autolysis and protein degradation. Whole organs should be perfused through arteries or veins with ice-cold phosphate buffered saline (PBS) to remove the blood that may confound data analysis. Additionally, connective tissue and fat, and other nontarget tissue, should be physically removed before processing. The organ tissue should then be minced prior to homogenization. For sections of tissue, blood can be removed by rinsing and mincing the tissue in ice- cold PBS.

2.2.2 Blood

Blood is a specialized body fluid consisting of plasma (fluid phase), erythrocytes, leukocytes, and platelets. Blood is collected by venipuncture using a needle and syringe. Anticoagulated blood can be centrifuged at approximately 3500 × g for 10 min to obtain a cell pellet (consisting of all cell types) and plasma. Anticoagulants include K2- and K3-ethylenediaminetetraacetic acid (EDTA), sodium citrate and heparin, and tubes containing these anticoagulants are commercially available. These anticoagulants can have various effects on the proteomic analysis (extensively examined in Rai et al., 2005). Plasma contains all elements of the blood fluid phase, including proteins used for coagulation. If blood is allowed to clot before centrifugation (i.e., no anticoagulant used), then the supernatant is serum, and the coagulation proteins have been removed. The use of serum versus plasma is an important decision, as they each yield different results in global proteomic analysis. There appears to be an increase in peptides (1–15 kDa in size) in serum, probably due to the effects of coagulation proteases (Omenn et al., 2005), which may interfere with discovery of disease biomarkers in that size range. Serum is more frequently archived, however, and therefore may provide a better source for retrospective studies. Either plasma or serum provides a rich source of potential biomarkers, as the blood circulates through all parts of the body, and may therefore carry disease markers from remote sites. However, it is important to note that while such potential biomarkers are likely to be present in plasma or serum, there will be significant dilution effects as compared with the site of perturbation. These diluted, low-abundance molecules may be difficult to pick out amid the background of high-abundance serum proteins such as albumin and immunoglobulin G (IgG). To circumvent this issue, the sam­ples need to be depleted of such high-abundance proteins. Additionally, whole blood can be simplified by the separation into individual cellular components (e.g., erythrocytes, leukocytes, platelets). Both of these strategies are described in more detail below.