Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods

256 Pages·2000·6.213 MB·English

by  T. Rabilloud, I. Humphery-Smith (auth.), Dr. Thierry Rabilloud (eds.)

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Springer-Verlag Berlin Heidelberg GmbH
Th. Rabilloud (Ed.)
Proteome Research:
Two-Dimensional
Gel Electrophoresis
and Identification Methods
With 45 Figures
Springer
Dr. THIERRY RABILLOUD
CEA-Laboratoire de Bioenergetique Cellulaire et Pathologique EA 2019,
DBMS/BECP
CEA -Grenoble, 17 rue des Martyrs
38054 Grenoble Cedex 9, France
ISBN 978-3-540-65792-7
Library of Congress Cataloging-in-Publication Data
Proteome research: two-dimensional gel electrophoresis and Identification methods / Th. Rabilloud
(ed.). p.cm. -(Principles and practice)
Includes bibliographical references and index.
ISBN 978-3-540-65792-7 ISBN 978-3-642-57105-3 (eBook)
DOI 10.1007/978-3-642-57105-3
1. Proteins-Analysis. 2. Gel electrophoresis. 1. Rabilloud, Th. (Thierry), 1961- . II, Series.
QP551.P7564 2000 572'.633--dc21
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© Springer-Verlag Berlin Heidelberg 2000
Originally published by Springer-Verlag Berlin Heidelberg New York in 2000
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Foreword
This book appears at a fascinating juncture in the history of the biomedical sci
ences. For the first time we can contemplate the possibility of preparing complete
molecular descriptions of living cells, including complete genomic sequence data,
lists and structures of the various RNAs, catalogues of all protein gene products
and their derivatives, maps showing the intracellular locations of each macro
molecule, and an index of all metabolites.
It is from this perspective that it is useful to examine the role of global high
resolution protein analysis, which is the topic of this book. A detailed analysis of
the proteins of living cells is an important activity, but can most of the informa
tion to be gained not be inferred from genomics? If the complete plans for an
organism, and all of the basic data required to express those plans are encoded in
DNA, can we not deduce all we need to know about cells from that information?
A long-term goal of biology must certainly be to attempt to characterize man
from genomic sequence data, as was suggested in the first proposal for the
human genome project (Anderson and Anderson 1985). However, this is not yet
possible, nor will it be in the foreseeable future. The complexity of living cells
defeats such efforts at present, and we must ask instead, what information is
required regarding the emerging field of proteomics which cannot now be
inferred from nucleic acid sequences or mRNA abundance data, and is unlikely
to be obtained in the foreseeable future?
It is now generally agreed that global protein analyses are necessary for the fol
lowing reasons: there is poor correlation between mRNA abundance and proteins
coded for (Tew et al. 1996; Anderson and Seilhamer 1997; Gygi et al. 1999);
almost all proteins are post-translationally modified; the passage of a gene prod
uct from site of synthesis to site of activity cannot always deduced from sequence
data; and function cannot be reliably inferred for all proteins (or fragments
thereof) from sequence information.
Hence, from a purely anatomical point of view, it is essential to do as complete
an analysis of the protein complement of individual cell types as possible, and
many of the chapters of this book address this objective.
However, if complete molecular inventories of cells become possible, including
detailed structural data and subcellular localizations, will that end our quest
regarding molecular anatomy? Will we understand the key differences between
living and non-living systems?
An essential aspect of biochemistry has been the reconstruction of processes
and pathways in vitro. These processes include glycolysis, respiration, protein
VI Foreword
synthesis, synthesis of DNA and RNA, and transport through isolated membrane
vesicles, among others.
We have postulated that no protein has a fixed rate of synthesis, but that all are
dynamically controlled and in a constant state of flux (Anderson and Anderson
1996), and that a cell may be considered to be a kanban or "just-in-time" system
with minimum excess inventory. At one extreme, one may postulate that most
components are grouped into a relatively small number of co-regulated sets, and
at the other, that the number of factors affecting the expression of each gene is so
large that each gene is effectively under separate control. Only by devising means
for up- and down-regulating individual genes experimentally can these alterna
tives be explored in detail.
Good quantitation of large sets of proteins is essential for one to begin to
answer these questions. When a given drug interacts with a target, is the target
up- or down-regulated, or unchanged? It has been assumed, largely on the basis
of in vitro studies where excess drug is present, that antisense compounds can
and do down-regulate protein production by hybridization arrest. However, stud
ies of intact animals have not clearly demonstrated down-regulation of a normal
cellular gene by an antisense pharmaceutical followed by a useful pharmacologi
cal effect. Systematic studies on global changes in protein abundance in
antisense-treated tissues remain to be done. Further, a great deal of work remains
to be done on the effects of known enzyme inhibitors on the abundance of the
inhibited enzyme in intact animals.
The most interesting question, however, concerns co-regulation and the
degree of interconnectedness between metabolic processes and pathways. Is it
possible to delete or inhibit any protein without affecting the abundance of oth
ers? The fact that many knockout mice survive deletions that whould be expected
to be fatal suggests a very large array of compensatory changes in gene expres
sion, which remain to be explored in detail. In such response arrays may lie many
of the answers to the question of what is unique about living systems. Only with
high-resolution, sensitive quantitative, and repeatable analyses which can be
done on very large numbers of samples (i. e., with complete automation) can
these questions be explored.
Unlike genomics, which involves the analysis of simple repeating polymers,
proteomics involves the study of very complex heteropolymers having a wide
range of sizes, physical properties, secondary and tertiary structures, solubilities,
and functions. Hence, proteomics is and will continue to be a much more diffi
cult field than genomics, will be more costly, and will require continued innova
tion and extensive support.
Genomics, for a range of organisms, now approaches completion. Proteomics,
and epigenomics (i. e., the science of how genomes and proteomes interact), are,
in contrast, only in their infancy. This book reviews the classes of interdisciplin
ary research required for this new science.
NORMAN G. ANDERSON
Large Scale Biology Corporation
Rockville, Maryland
Foreword VII
References
Anderson NG, Anderson NL (1985) A policy and program for biotechnology. American Biotechnol
ogy Laboratory, Sept/Oct 1985
Anderson NG, Anderson NL (1996) Twenty years of two-dimensional electrophoresis: past, present
and future. Electrophoresis 17:443-453
Anderson NL, Seilhammer J (1997) A comparison of selected mRNA and protein abundances in
human liver. Electrophoresis 18:533-537
Gygi SP, Rochon Y, Franza BR, Aebersold R (1999) Correlation between protein and mRNA abun
dance in yeast. Mol Cell Biol1999 19:1720-30
Tew KD, Monks A, Barone L, Rosser D, Akerman G, Montali JA, Wheatly JB, Schmidt DE Jr (1996)
Glutathione-associated enzymes in the human cell lines of the National Cancer Institute Drug
Screening Program. Mol Pharmacol50:149-159
Contents
1 Introduction: The Virtue of Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
THIERRY RABILLOUD and IAN HUMPHERY SMITH
2 Solubilization of Proteins in 2D Electrophoresis .................... 9
THIERRY RABILLOUD and MIREILLE CHEVALLET
1 Introduction................................................... 9
2 Rationale of Solubilization-Breaking Molecular Interactions. . . . . . . . . .. 10
3 Initial Solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 14
4 Solubility During IEF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 22
5 Conclusions: Current Limits and How to Push Them ................ 24
References ..................................................... 27
3 Two-Dimensional Electrophoresis with Carrier Ampholytes .......... 31
CHRISTELLE MONRIBOT and HELIAN BOUCHERIE
1 Introduction................................................... 31
2 Sample Preparation ............................................. 36
3 First Dimension. Standard Isoelectric Focusing ..................... 39
4 Second Dimension. Standard Slab Gel Electrophoresis ............... 43
5 Modification of the Standard Two-Dimensional Gel Method .......... 47
6 Visualization of Separated Proteins: Silver Staining .................. 51
References ..................................................... 51
Appendix: Problems and Troubleshooting .......................... 53
4 Two-Dimensional Electrophoresis with Immobilized pH Gradients .... 57
ANGELIKA GORG and WALTER WEISS
1 Introduction................................................... 57
2 Sample Preparation ............................................. 65
3 First Dimension: IEF with IPGs ................ . . . . . . . . . . . . . . . . . .. 69
4 Equilibration of IPG Strips ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 80
5 Second Dimension: SDS-PAGE .................................... 82
References ..................................................... 91
Appendix A: General Troubleshooting ............................. 93
Appendix B: Troubleshooting for IPG and Horizontal PAGE. . . . . . . . . .. 99
x Contents
5 Detection of Proteins on Two-Dimensional Electrophoresis Gels ...... 107
THIERRY RABILLOUD and STEPHANE CHARMONT
1 Introduction................................................... 107
2 Detection by Organic Dyes ....................................... 107
3 Detection by Differential Precipitation of Salts ...................... 111
4 Detection by Metai Ion Reduction (Silver Staining) .................. 113
5 Detection by Fluorescence ....................................... 119
6 Detection of Radioactive Isotopes ................................. 123
7 Conclusions and Future Prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 124
References ..................................................... 125
6 Blotting and Immunoaffinity Identification of Two-Dimensional
Electrophoresis-Separated Proteins ............................... 127
BARBARA MAGI, LUCA BINI, BARBARA MARZOCCHI,
SABRINA LIBERATORI, ROBERTO RAGGIASCHI and VITALIANO PALLINI
1 Introduction................................................... 127
2 Protein Transfer onto Membranes ................................. 128
3 Membrane Staining ............................................. 132
4 Immunodetection ............................................... 134
5 Assignment of Two-Dimensional Immunoreactive Spots by Matching .. 137
References ..................................................... 139
7 Identification of Proteins by Amino Acid Composition
After Acid Hydrolysis ........................................... 143
MARGARET I. TYLER and MARC R. WILKINS
1 Introduction ................................................... 143
2 Blotting of Proteins from Gels to PVDF Membranes ................. 144
3 Hydrolysis of PVDF-bound Proteins ............................... 145
4 Extraction of Amino Acids from PVDF Membranes .................. 148
5 Derivatization and Chromatography ............................... 149
6 Amino Acid Analysis Troubleshooting Guide ....................... 152
7 Protein Identification by Database Matching ........................ 155
8 Identification by N -Terminal Sequence Tags and Amino Acid
Composition ................................................... 157
9 Conclusions.................................................... 159
References ..................................................... 159
8 Identification by Amino Acid Composition Obtained from Labeling. .. 163
JEAN LABARRE and MICHEL PERROT
1 Introduction ................................................... 163
2 Choice and Labeling Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 164
3 Single Labeling Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 167
4 Double Labeling Method Based on 35S Decay ....................... 167
5 Double Labeling Method using Scintillation Counting. . . . . . . . . . . . . . .. 170
6 Determination of pI and Mr ...................................... 172
7 Construction of the Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 173
8 Search in a Protein Database ..................................... 173