proteomics in practice - a laboratory manual of proteome analysis - tom naven

Reiner Westermeier, Tom Naven Proteomics in Practice Proteomics in Practice: A Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Copyright  2002 Wiley-VCH Verlag GmbH ISBNs: 3-527-30354-5 (Hardcover); 3-527-60017-5 (Electronic) Related title from Wiley-VCH Reiner Westermeier Electrophoresis in Practice Third Edition ISBN 3-527-30300-6 Proteomics in Practice A Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Proteomics in Practice: A Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Copyright  2002 Wiley-VCH Verlag GmbH ISBNs: 3-527-30354-5 (Hardcover); 3-527-60017-5 (Electronic) Dr. Reiner Westermeier Dr. Tom Naven Amershan Biosciences Europe GmbH Munzinger Str. 9 79111 Freiburg Germany & This book was carefully produced. Never- theless, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inad- vertently be inaccurate. Library of Congress Card No.: applied for British LibraryCataloguing-in-PublicationData: A catalogue record for this book is available from the British Library. Die Deutsche Bibliothek ± CIP Cataloguing- in-Publication Data: A catalogue record is available from Die Deutsche Bibliothek.  Wiley-VCH Verlag-GmbH Weinheim, 2002 All rights reserved (including those of translation in other languages). No part of this book may be reproduced in any form ± by photoprinting, microfilm, or any other means ± nor transmitted or translated into a machine language without written permission from the publisher. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. printed in the Federal Republic of Germany printed on acid-free paper. 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KG, Grünstadt ISBN 3-527-30354-5 Proteomics in Practice: A Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Copyright  2002 Wiley-VCH Verlag GmbH ISBNs: 3-527-30354-5 (Hardcover); 3-527-60017-5 (Electronic) V Preface IX Foreword XI Abbreviations, symbols, units XIII Glossary of terms XVII Part I: Proteomics Technology 1 1 Introduction 1 1.1 Applications of proteomics 6 1.2 Separation of the protein mixtures 6 1.3 Detection 7 1.4 Image analysis 7 1.5 Identification of proteins 8 1.6 Characterization of proteins 9 1.7 Functional proteomics 9 2 Expression proteomics 11 2.1 Two-dimensional Electrophoresis 11 2.1.1 Evolution of the 2-D methodology 12 2.1.2 Sample preparation 15 2.1.3 First Dimension: Isoelectric focusing 27 2.1.4 Second dimension: SDS-PAGE 57 2.1.5 Detection of protein spots 79 2.1.6 Image analysis 87 2.2 Spot handling 97 2.2.1 Spot cutting 98 2.2.2 Protein cleavage 100 2.3 Mass spectrometry 108 2.3.1 Ionisation 112 2.3.2 Ion separation 118 Contents Proteomics in Practice: A Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Copyright  2002 Wiley-VCH Verlag GmbH ISBNs: 3-527-30354-5 (Hardcover); 3-527-60017-5 (Electronic) VI 2.3.3 Tandem mass spectrometry (MS/MS) 125 2.4 Protein identification by database searching 135 2.4.1 Peptide mass fingerprint 136 2.4.2 Peptide mass fingerprinting combined with composition information 138 2.4.3 Peptide mass fingerprint combined with partial sequence information 139 2.4.4 Product ion MS/MS sequence data 143 2.4.5 De novo sequencing 145 2.5 Methods of proteome analysis 154 2.5.1 2D-MS 154 2.5.2 LC-MS/MS 157 2.5.3 Quantitative proteomics 159 Part II Course Manual 161 Equipment, Consumables, Reagents 163 Step 1: Sample preparation 169 1 Stock solutions 170 2 Examples 172 3 Microdialysis 175 4 Precipitation 177 5 Basic proteins 181 6 Very hydrophobic proteins 182 7 Quantification 184 8 SDS samples for HMW proteins separation 186 Step 2: Isoelectric focusing 187 1 Reswelling tray 188 2 Rehydration loading and IEF in IPGphor strip holders 190 3 IEF in the cup loading strip holder (rehydration loaded strips) 192 4 Cup loading IEF 194 5 Staining of IPG strips 196 Step 3: SDS Polyacrylamide Gel Electrophoresis 199 1 Casting of SDS polyacrylamide gels 199 1.1 Stock solutions 199 1.2 Cassettes for laboratory made gels 201 1.3 Multiple Gel Caster (up to 14 gels) 203 1.3.2 Homogeneous gels 204 1.3.3 Gradient gels 207 1.4 Gel Caster for up to six gels 212 2 Inserting ready-made gels into cassettes 219 3 Preparation of the SDS electrophoresis equipment 222 Contents VII 3.1 Stock solutions for the running buffers 222 3.2 Setting up the integrated high-throughput instrument 223 3.3 Setting up the six gel instrument 223 4 Equilibration of the IPG strips and transfer to the SDS gels 224 4.1 Equilibration 224 4.2 Application of the IPG strips onto the SDS gels 224 4.3 Application of molecular weight marker proteins and 1-D samples 226 4.4 Seal the IPG strip and the SDS gel 226 5 The SDS electrophoresis run 227 5.1 The integrated high-throughput instrument 227 5.2 The six-gel instrument with standard power supply 229 Step 4: Staining of the gels 233 1 Colloidal Coomassie Brilliant Blue staining 233 1.1 Solutions 233 1.2 Staining 234 2 Hot Coomassie Brilliant Blue staining 234 3 Silver staining 235 4 Fluorescence staining 238 5 Preserving and drying of gels 238 Step 5: Scanning of gels and image analysis 239 1 Scanning 239 1.1 Gels stained with visible dyes 239 1.2 Scanning fluorescent dyes 241 2 Spot detection and background parameters 241 2.1 Automated spot detection 241 2.2 Spot filtering 242 2.3 Spot editing 242 2.4 Background correction 243 3 Evaluation of 2-D patterns 243 3.1 Reference gel 243 3.2 Automatic gel matching 244 3.3 Normalisation 245 3.4 pI and M r calibration 245 3.5 Difference maps 246 3.6 Averaged gels 247 3.7 Quantification 247 3.8 Creating reports 247 3.9 Spot picking 247 Step 6: Fluorescence difference gel electrophoresis 249 1 Preparing a cell lysate compatible with CyDye labelling 250 2 Reconstituting the stock CyDye in Dimethylformamide (DMF) 251 3 Preparing CyDye solution used to label proteins 252 Contents VIII Step 7: Spot excision 255 Step 8: Sample destaining 259 Step 9: In-gel digestion 261 Step 10: Microscale desalting and concentration of sample 263 Step 11: Chemical derivatisation of the peptide digest 265 Step 12: MS analysis 269 Step 13: Calibration of the MALDI-ToF MS 273 Step 14: Preparing for a database search 277 Step 15: PMF database search unsuccessful 281 A Trouble shooting 283 1 Two-dimensional Electrophoresis 283 1.1 Isoelectric focusing in IPG strips 283 1.2 SDS PAGE 285 1.3 Staining 286 1.4 DIGE fluorescence labelling 287 1.5 Results in 2-D electrophoresis 288 2 Mass spectrometry 291 References 295 Index 311 Contents The objective of Proteomics in Practice is to provide the reader with a comprehensive reference and manual guide for the successful analy- sis of proteins by 2-D electrophoresis and mass spectrometry. The idea for the book has come from the continuing success and favoura- ble responses received from the scientific public for our on-going pro- teomics seminar and practical courses we have delivered in the past twelve months. The book will include a theoretical introduction, comprehensive practical section complete with worked examples, a unique troubles- hooting section designed to answer many of the frequently asked questions regarding proteome analysis and a thorough reference list to guide the interested reader to further detail. The theoretical section will introduce the fundamentals behind the techniques currently being used in proteomics today and describe how the techniques are used for proteome analysis. However, the practical aspects of the book will not address many of these methods, but will instead focus on the main stream methodolo- gy of 2-D electrophoresis and mass spectrometry. 2-D electrophoresis is still the most successful method of resolving a proteome with increasing reproducibility and automation. All aspects for the suc- cessful performance of 2-D electrophoresis and image analysis will be addressed in practical detail. Subsequently, the importance of mass spectrometry, sequence databases and search engines for successful protein identification will be discussed. The practical section of the book is in principle a course manual, which has been optimized over a number of years. The success of the ªElectrophoresis in Practiceº book range, has demonstrated that a course manual is a useful guide for daily work in the laboratory. The section will describe how to achieve good, reliable and reproducible results using a single instru- mental setup, instead of presenting a wide choice of techniques and instruments. In this book some statements may be found, which do not comply with the ªhigh endº technological achievements pub- IX Preface Proteomics in Practice: A Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Copyright  2002 Wiley-VCH Verlag GmbH ISBNs: 3-527-30354-5 (Hardcover); 3-527-60017-5 (Electronic) Preface lished. The experimental procedures are restricted to the area of robustness and routinely achievable good results. The authors understand and wholly appreciate that the analysis of post-translational modifications such as phosphorylation and glycosy- lation is an integral aspect of proteomics. As such the theoretical, technical and practical issues involved will be addressed in great detail in a subsequent edition. Approaches for functional proteomics are still varying and many procedures are under development. These methods will be added in a later edition. As the technical developments in this field are proceeding so fast, the contents of the book need to be updated every few months. The reader can have access to a web-site at WILEY-VCH: http://www3.in- terscience.wiley.com/XXXXXX, which will contain the updated chap- ters and recipes. Reiner Westermeier Tom Naven January 2002 Thanks to: Jan Axelsson, Tom Berkelman, Philippe Bogard, Josef Bülles, Maria Liminga, Tom Keough, Matrixscience.com, Staffan Renlund, Günter Thesseling. X [...]... which has opened the door for proteomics by providing a sequencebased framework for mining the human proteome and that of other organisms It is evident that proteomics has attracted a substantial following, with an influx of investigators and of biotechnology and pharmaceutical companies that are taking an active interest in the field, as well as an influx of a new generation of scientists in training... current Sam Hanash MD, PhD Department of Pediatrics A 520 MSRBI University of Michigan Ann Arbor MI 48109 USA Proteomics in Practice: A Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Copyright  2002 Wiley-VCH Verlag GmbH ISBNs: 3-5 2 7-3 035 4-5 (Hardcover); 3-5 2 7-6 001 7-5 (Electronic) Abbreviations, symbols, units 1-D electrophoresis 2-D electrophoresis A A,C,G,T AEBSF API APS AU 16-BAC.. .Proteomics in Practice: A Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Copyright  2002 Wiley-VCH Verlag GmbH ISBNs: 3-5 2 7-3 035 4-5 (Hardcover); 3-5 2 7-6 001 7-5 (Electronic) Foreword Proteomics is in an extraordinary growth phase This is due to a great extent to the fact that the major undertaking of sequencing the human and other important genomes has largely been accomplished,... matrix into more reactive species, which can add to the polypeptide Can also result from salt ions, Na+ etc., that are embedded in the matrix Proteins are loaded in amounts of 10 to 100 lg Mostly broad pH intervals are used in the first dimension The mass of a molecule of a given empirical formula calculated using the average atomic weights for each element An average mass is obtained in MALDI-TOF-MS... quadrupole, time -of- flight and magnetic sector The area of interest to be measured in an experiment Or the capability of the analyser An instrument that measures the mass to-charge ratio (m/z) of ionized atoms or molecules Comprises three parts: an ion source, a mass analyser, and an ion detector A technique for analysing the molecular weight of molecules based upon the motion of a charged particle in an electric... alkylation 75 amino acid modification 278 amino acid sequence 79 ammonium bicarbonate 264 amphoteric buffers 15 amphoteric molecules 27 analytical applications 19 anodal buffer 222 application point 51 automated image analysis 96 sample preparation 27 automated stainer 236 averaged gels 247 average mass 279 averaging gels 90, 95 b b-ion 128 b-ion series 127, 147 backed gels 88 background 35 subtraction, correction... capacity of a standard laboratory equipment The fast acquisition of the human genome was only possible by the application of an industrial approach Exactly the same happens now for proteome analysis: most of the data will certainly be delivered by Proteomics factoriesº Because of the complexity of the sample, two-dimensional polyacrylamide gel electrophoresis has been widely utilized as the standard... crystallization Method Tandem mass spectrometry (MS/MS) Thin layer method Threshold fluence Time-ion extraction (time lag focussing or delayed extraction) Time -of- flight (TOF) analyser Tuning Two-dimensional electrophoresis Unit resolution Proteomics in Practice: A Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Copyright  2002 Wiley-VCH Verlag GmbH ISBNs: 3-5 2 7-3 035 4-5 (Hardcover); 3-5 2 7-6 001 7-5 ... which allow the analysis of proteins and peptides with high sensitivity, accuracy and throughput Online peptide fragmentation allows quick amino acid sequence analysis of low amounts of peptides at low running cost Also the analysis of post-translational modification can be addressed using this technology Genomics: Thanks to the development of high throughput DNA sequencing genomic databases of many... identification, with which the field of proteomics has been tightly associated in the past decade Evidently, 2-D gels have come under assault lately, due in part to the influx of new investigators to the field, most of whom have no particular leanings towards 2-D gels and consider the lack of automation and the limited sensitivity of 2-D gels as major drawbacks While XI XII Foreword non-2-D gel based approaches . Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Proteomics in Practice: A Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Copyright  2002 Wiley-VCH Verlag. spectrum A plot of ion abundance (y-axis) against mass-to- charge ratio (x-axis). Mass-to-charge ratio (m/z) A quantity formed by dividing the mass of an ion (in Da units) by the number of charges carried. technological achievements pub- IX Preface Proteomics in Practice: A Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Copyright  2002 Wiley-VCH Verlag GmbH ISBNs: 3-5 2 7-3 035 4-5 (Hardcover);