Methods in Animal Proteomics

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Section 1 Exploring Animal Proteomes

1 An Introduction to Animal Proteomics Phillip D. Whitfi eld 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, refl ecting the true biochemical outcome of genetic informa-tion. However, proteomics has developed from cataloguing proteins to an advanced discipline that requires a substantial investigation of the protein world, defi ning 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 profi le. 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 emerg-ing fi eld of animal proteomics. It is by no means exhaustive but is aimed at capturing the excitement of current practitioners of the fi eld and relates to their experiences. The book addresses the experimental strategies and tech-niques employed in animal proteomics studies. It also outlines key applica-tions of proteomics to the study of animal systems across a variety of disciplines. Importantly, the focus of the book extends beyond the use of

Methods in Animal Proteomics, First Edition. Edited by P. David Eckersall, Phillip D. Whitfi eld.© 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc.

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laboratory rodent models and instead encompasses a broad range of compan-ion and production animals, birds, fi sh, reptiles, and wildlife.

1.2 Exploring Animal Proteomes

Proteomics has the ability to encompass the large - scale identifi cation, char-acterization, and quantifi cation of the proteins in animal systems. The advances made in proteomics have been underpinned by signifi cant technical develop-ments, which have revolutionized protein analysis. The investigation of animal proteomes requires a combination of effi cient 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 signifi cant practical and analytical challenges.

There is a wide range of biofl uids and tissue samples that can be been employed in animal proteomic experiments. These include plasma, serum, blood cells, urine, cerebrospinal fl uid, amniotic fl uid, synovial fl uid, seminal fl uids, bile, feces, saliva, milk, eggs, wool, and venom as well as many dif-ferent tissue types, which refl ect 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 fl uids 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 meth-odologies to be used in the analysis of the protein complement.

A variety of powerful experimental approaches exist for profi ling of animal proteomes. In Chapter 3 Ingrid Miller provides a comprehensive review of the principles and technical aspects relating to electrophoretic and chromato-graphic techniques that are routinely used for protein separation and isolation. The chapter also outlines how these tools may be employed to discover pro-teins that are differentially expressed in animal systems. In proteomic experi-ments proteins are typically identifi ed 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

AN INTRODUCTION TO ANIMAL PROTEOMICS 7

of interest, either in - solution or excised from a gel, are digested with a pro-teolytic enzyme, typically trypsin, and the resultant peptides are analyzed by mass spectrometry. A process referred to as peptide mass fi ngerprinting (PMF) utilizes the capability of matrix - assisted laser desorption/ionization - time of fl ight - mass spectrometry (MALDI - TOF - MS) to produce a unique pattern of peptide ions for individual proteins. These proteins are then identifi ed 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 diffi culty with this approach is the lack of complete and anno-tated 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 pro-teins in animals where little or no sequence information exists, although it still relies on suffi cient sequence information being available from a homolo-gous 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 confi -dence in the protein identifi cations provided by a typical proteomic experi-ment 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 multidi-mensional protein identifi cation 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 poly-acrylamide gel electrophoresis (1 - D SDS - PAGE) coupled with LC - MS/MS have also been developed as part of strategies aimed at the routine qualitative identifi cation of proteins (Schirle et al., 2003 ).

While the high - throughput nature of shotgun proteomics approaches has gained signifi cant popularity, it should be noted that protein identifi cation by this method is still very challenging. From a single experiment large amounts of data are generated, which must be assembled to give protein identifi cation.

8 EXPLORING ANIMAL PROTEOMES

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 identifi ca-tions. In addition, insuffi cient protein sequence coverage and sequence redun-dancy, 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 unidentifi ed, which has signifi cant implications for biological interpretation of proteomic data.

Novel mass proteomic approaches are also now emerging to enhance protein identifi cation by characterizing specifi c 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 subject-ing 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 confi dent protein assignment from a single peptide. Many of these bioinformatic issues associated with identifi cation of proteins from animal body fl uids and tissues are discussed by Blakeley and colleagues in Chapter 5 .

Experimental approaches to compare protein profi les 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 defi ned 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 defi ne 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 fi elds 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

AN INTRODUCTION TO ANIMAL PROTEOMICS 9

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 mecha-nisms. 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 pharma-cological activities (Escoubas and King, 2009 ).

The ability to obtain a profi le 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 tech-nologies 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 man-agement of livestock, poultry, and fi sh, 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 refl ected 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 deter-mine the host ’ s response to infection, investigate the mechanisms of transmis-sion 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 col-leagues (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 defi ne meat and fi sh 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 intro-ductory text for animal scientists with little or no experience of proteomics, while providing an up - to - date reference for researchers with a background in

10 EXPLORING ANIMAL PROTEOMES

the area. We hope that readers will fi nd the book interesting and that it proves to be a useful source of information for anyone working in the growing fi eld of animal proteomics.

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