Proteome Research: New Frontiers in Functional Genomics PDF
Preview Proteome Research: New Frontiers in Functional Genomics
To Sobet, Brynnie, Catherine and Anne-Catherine To our children To our parents To Alan Williams and Matthieu Funk who would have enjoyed this had they stayed around to experi ence it "'tJ :-lJ Z n "'tJ r m til » z o "'tJ »:lJ n ---I n m Springer-Verlag Berlin Heidelberg GmbH M.R. Wilkins . K.L. Williams' R.D. Appel • D.F. Hochstrasser (Eds.) Proteome Research: New Frontiers in Functional Genomics With 36 Figures, 16 in Color Springer DR. MARC R. WILKINS DR. RON D. ApPEL Hopitaux Universitaires de Geneve Hopitaux Universitaires de Geneve et Universite de Geneve Division d'Informatique Medicale Laboratoire Central de Chimie Clinique 24, rue Micheli-du-Crest 24, rue Micheli-du-Crest 1211 Geneve 14 1211 Geneve 14 Switzerland Switzerland PROF. KEITH L. WILLIAMS PROF. DENIS F. HOCHSTRASSER Australian Proteome Analysis Facility Hopitaux Universitaires de Geneve andMUCAB et Universite de Geneve Macquarie University Laboratoire Central de Chimie Clinique School of Biological Sciences 24, rue Micheli-du Crest Sydney 1211 Geneve 14 NSW 2109 Australia Switzerland ISBN 978-3-540-62753-1 ISBN 978-3-662-03493-4 (eBook) DOI 10.1007/978-3-662-03493-4 CIP Data applied for Die Deutsche Bibliothek -CIP-Einheitsaufnahme Proteome research : new frontiers in functional genomics / ed. Marc R. Wilkins ... -Berlin; Heidelberg ; New York; Barcelona; Hong Kong; London ; Milan ; Paris; Santa Clara; Singapore; Tokyo: Springer 1997 (Principles and practice) ISBN 3-540-62775-8 This work is subject to copyright. AlI rights are reserved, whether the whole or part of the material is concerned, specifica11y the rights of translation, reprinting, reuse of illustrations, recitation, broadca sting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permissions for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. © Springer-Verlag Berlin Heidelberg 1997 Originally published by Springer-Verlag Berlin Heidelberg 1997 The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Camera ready by the editors Cover design: D&P, Heidelberg using the illustration HepG2 2-D gel reference map from the SWISS-2DPAGE database. SPIN 10571786 39/3137 5 4 3 2 1 0-Printed on acid free paper Acknowledgments This book is the fruit of a fantastic collaboration and friendship established around the world and in particular between scientists from Australia, Italy and Switzer land. We met in Sydney, Siena and Geneva, exchanged researchers and in 1996 at the Siena Proteome meeting conceived this book on Proteomics. There followed a division of duties that has put a heavy load on the researchers in our respective lab oratories. All of us are busy people and we acknowledge the great contributions which our authors have made against tight deadlines! Proteomic developments in Sydney have been possible thanks to the financial support of Macquarie University, the New South Wales State Government and the Federal Australian Government who together helped establish the first national proteome facility, the Australian Proteome Analysis Facility (APAF) in 1996. We also acknowledge the Australia Research Council and Australian Medical Research Council for supporting our research which has borne fruit as the pro teome. The Sydney group acknowledges the unstinting support by many compa nies involved in scientific instrument developments and diagnostics. Without the support of Beckman Instruments (Australia and U,S.A.), Bio-Rad Laboratories (Australia and U.S.A.), GBC Scientific Equipment, Gradipore Limited, Hewlett Packard, Amrad Pharmacia Biotech, there would be no prototype instruments in our laboratory. Proteomic developments in Geneva for the last fifteen years have been possible thanks to the financial support of the University of Geneva and University Hospital of Geneva, the Inter Maritime, Michelham, Montus and Helmut Horten Founda tions, the Swiss National Fund for Scientific Research and several world-wide companies such as Electrophoretics pIc, Bio-Rad Laboratories and Oxford GlycoSciences. The Geneva group also thanks Professors Alex F. Muller, Jean Raoul Scherrer, Christian Pellegrini, Francis Waldvogel, Alain Junod and Robin Offord who have trusted our approach and supported our work for many years. The first two proteome meetings in Siena in 1994 and 1996, which stimulated the realisation of this book, were possible thanks to Electrophoretics pIc and the Uni versity of Siena. Finally, we would like to thank Professor Edmond Fischer for his most support ive preface, which is the best encouragement we can get to further pursue our pro teome work. Marc Wilkins, Keith Williams, Ron Appel and Denis Hochstrasser Preface This is a timely book. It is being published as all the information stored in our DNA is about to be revealed, but demonstrates convincingly why this accumulated nucleic acid-based knowledge can only take us so far. The human genome might enable us to predict the proteins that can potentially be generated, but not where and when or at what level. It cannot tell us the cells in which proteins will be expressed or at which stages of development or differentiation this will happen. Nor can it take into account the enormous diversification of structure that results from alternate splicing, gene insertion, switching by deletions and recombination and other kinds of rearrangements as seen, for instance, in the immune system. Gene structure alone tells us very little about the physiological function of proteins since it ignores the co- and post-translational modifications to which they are sub jected, such as their processing by limited proteolysis, their glycosylation, prenyla tion, ADP-ribosylations etc., and, of course, reversible phosphorylation. The regulation of cellular processes requires a myriad of commands, positive and negative, to keep under control all reactions that take place and to make sure that no crucial event occurs at an inappropriate time or place. And we know today that the predominant signal that orchestrates these reactions, that turns the switches on and off, relies on protein phosphorylation. This is present to such an extent that at least 30% of all the proteins found in a mammalian cell extract exist in a phosphor ylated state, even though one wouldn't know whether all these phosphorylation reactions are physiologically significant. But even if we had all this knowledge at hand, one wouldn't know how cells react to given signals or, in multicellular organisms, synchronise their behaviour in response to internal or external demands. The modified proteins that are ultimately generated are only the words used by cells to transduce their signals or communi cate with one another. For sentences to be constructed or for cross-talks to be initi ated, proteins must interact with target, adapter or docking proteins and cells must interact with cells. This requires a plethora of binding motifs (e.g. the src homology SH2 and SH3 domains, or the PH, WW, PDZ domains), some no longer than a few amino acid residues, and further special sequences that determine the localisation of proteins to subcellular elements, direct their translocation in or out of the nucleus, their insertion into the endoplasmic reticulum or the plasma membrane or target them for destruction after internalisation. Cell-cell interaction brings into play an intricate, combinatorial system of cell adhesion molecules, crucial to the x establishment of such highly sophisticated networks of communication one finds, for instance, in the immune system or the far more complex central nervous system where more than a billion cells speak with one another through a million billion synapses, ultimately leading to the generation of thought, memory and conscious ness. In all, then, no matter how distinct proteins might be from one another, they must be viewed as mosaics of structural elements, sorts of necklaces made up of a vari ety of beads in which each bead carries its own characteristic properties. However, it is the sum of these beads, their choice and particular disposition within the pep tide chain that will ultimately determine the overall architecture and function of a protein. This book, written and edited by those who conceived the notion of proteomics and contributed the most to its development, is the first to offer a comprehensive perspective of the field. It describes authoritatively its origin, fundamentals and background; its methodologies and how the data collected should be analysed, stored, retrieved and applied by the research and industrial scientist as well as the clinician. It eloquently documents how far these new technologies have taken us and where they might lead us in the future. June 19, 1997 Edmond H. Fischer Prof. Emeritus of Biochemistry University of Washington 1992 Nobel Laureate in Medicine Contents 1 Introduction to the Proteome .................................. 1 Keith L. Williams and Denis F. Hochstrasser 1.1 Proteome: a new word, a new field of biology ...................... 1 1.2 The proteome and technology ................................... 5 1.2.1 Thinking in two dimensions .................................... 6 1.2.2 Further dimensions in protein analysis ............................ 8 1.2.3 Information and the proteome ................................... 9 1.3 Looking towards new frontiers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Acknowledgments .......................................... 11 References ................................................. 11 2 Two-Dimensional Electrophoresis: The State of the Art and Future Directions ....................................... 13 Ben R. Herbert, Jean-Charles Sanchez and Luca Bini 2.1 Introduction ................................................ 13 2.2 Sample preparation .......................................... 15 2.2.1 Increasing protein solubility with chaotropes and surfactants ......... 15 2.2.2 The choice of reducing agent .................................. 16 2.2.3 Removal of nucleic acids ..................................... 16 2.3 Sample loading on IPG gels ................................... 18 2.3.1 Sample application during IPG rehydration ....................... 18 2.4 Low abundance proteins ...................................... 19 2.4.1 Cell fractionation and protein prefi"actionation ..................... 20 2.4.2 High protein loads combined with narrow pH gradients ............. 21 2.4.3 Sensitive detection .......................................... 22 2.5 Basic proteins .............................................. 24 2.5.1 Separation of basic proteins on IPG gels ......................... 24 2.6 Protein quantitation .......................................... 25 2.7 Future directions for 2-D PAGE ................................ 26 2.7.1 Simplifying the IPG-SDS-PAGE interface ........................ 27 2.7.2 Fluorescent protein detection .................................. 28 2.7.3 High throughput 2-D PAGE ................................... 28 2.8 Conclusion ................................................ 29 Acknowledgments .......................................... 29 References ................................................. 30 XII 3 Protein Identification in Proteome Projects ..................... 35 Marc R. Wilkins and Andrew A. Gooley 3.1 The purpose of protein identification ............................ 35 3.2 An overview of protein identification strategies .................... 36 3.3 Primary attributes for protein identification ....................... 38 3.3.1 Protein species of origin ...................................... 38 3.3.2 Protein isoelectric point ...................................... 38 3.3.3 Protein apparent mass and mass ................................ 40 3.3.4 Protein N-and C-terminal sequence tags ......................... 41 3.3.5 Extensive N-terminal protein sequence .......................... 45 3.4 Secondary attributes for protein identification ..................... 46 3.4.1 Attributes of pep tides from mass spectrometry .................... 46 3.4.2 Protein amino acid composition ................................ 54 3.5 Cross-species protein identification ............................. 56 3.6 Future developments and conclusions ........................... 60 Acknowledgments .......................................... 60 References ................................................. 61 4 The Importance of Protein Co-and Post-Translational Modifications in Proteome Projects ........................... 65 Andrew A. Gooley and Nicolle H. Packer 4.1 Introduction ................................................ 65 4.2 An overview of modifications: what are they and where do they occur? 68 4.3 Modifications that influence protein charge on 2-D PAGE ........... 70 4.4 Analysis of co- and post-translational modifications after 2-D PAGE ... 70 4.4.1 Presentation of the modified protein ............................. 70 4.4.2 Detection of co-and post-translational modifications ............... 74 4.4.3 Analysis of co- and post-translational modifications ................ 76 4.4.4 Mass spectrometry perspectives in the analysis of protein co- and post-translational modifications ................................ 83 4.5 Future directions ............................................ 86 Acknowledgments .......................................... 86 References ................................................. 86 5 Proteome Databases ........................................ 93 Amos Bairoch 5.1 Introduction ................................................ 93 5.2 Protein sequence databases .................................... 93 5.2.1 SWISS-PROT .............................................. 95 5.2.2 TrEMBL ................................................. 100 5.2.3 Completeness and non-redundancy issues ....................... 100 5.2.4 Specialised protein sequence databases ......................... 102 5.3 Nucleotide sequence databases ................................ 103 5.3.1 Hidden treasures ........................................... 107 5.4 Pattern and profile databases ................................. 108
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