2-D Proteome Analysis Protocols

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Polyamine Protocols, edlted by DavrdM L Morgan, 1998 Antibaeterial Peptide Protocols, e&led by Wham M Shafer, 1997 Protein Synthesa: Methods and Protocols, edited by Robm Marlm. 1998 Glycoanalysis Protocols (2nd. ed.), edlted by Ehzaberh F Howell, 1998

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cDNA Llbrary Protocols, edlted by /an G Cowell and Carohne A Auslm, 1997 Gene Isolation and Mapping Protocols, edited by Jacquehne Boultwood, 1997

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66 65 64 63

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Humana Press Totowa, New Jersey

M E T H O D S I N M O L E C U L A R B I O L O G Y TM

Edited by

Andrew J. LinkDepartment of Molecular Biotechnology,

University of Washington, Seattle, WA

2-D ProteomeAnalysis

Protocols

© 1999 Humana Press Inc.999 Riverview Drive, Suite 208Totowa, New Jersey 07512

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2-D proteome analysis protocols / edited by Andrew J Link.p. cm. —(Methods in molecular biology : vol. 112)

Includes index.ISBN 0-89603-524-7 (alk. paper)1. Protein—Analysis. 2. Gel electrophoresis. 3. Gene expression—Research—Methodology. I.Link, Andrew J. II. Series: Methods in molecular biology (Totowa, N. J.); 112.QP551.A14 1999 98-38677572'.6—dc21 CIP

Preface

v

With the completion of sequencing projects and the advancement of ana-lytical tools for protein identification, proteomics—the study of the expressedpart of the genome—has become a major region of the burgeoning field offunctional genomics. High-resolution 2-D gels can reveal virtually all pro-teins present in a cell or tissue at any given time, including posttranslationallymodified proteins. Changes in the expression and structure of most cellularproteins caused by differentiation or external stimuli can be displayed andeventually identified using 2-D protein gels.

2-D Proteome Analysis Protocols covers all aspects of the use of 2-Dprotein electrophoresis for the analysis of biological problems. The contribu-tors include many of the leaders in the fields of biochemistry and analyticalchemistry who were instrumental in the development of high-resolution 2-Dgels, immobilized pH gradients, computer analysis, and mass spectrometry-based protein identification methodologies.

This book is intended as a benchtop manual and guide both for novicesto 2-D gels and for those aficionados who wish to try the newer techniques.Any group using protein biochemistry—especially in the fields of molecularbiology, biochemistry, microbiology, and cell biology—should find this bookeminently useful.

2-D Proteome Analysis Protocols takes the researcher through the com-plete process of working with 2-D protein gels from making the protein ex-tract to finally identifying the proteins of interest. It includes protocols forgenerating 2-D protein extracts from most of the standard model organisms,including bacteria, yeast, nematode, Drosophila, plants, mouse, and human.This book covers the traditional methods of using carrier ampholytes in thefirst dimension and the growing movement in the field toward immobilizedpH gradients. A brief description of the advantages and disadvantages of eachmethod is given. Analytical and preparative 2-D gels, including the lastestprotocols for casting IPG gradients and in-gel rehydration of IPG strips, arecovered. For the second dimension, methods for running flatbed or verticalgels, including homogeneous and gradient gels, are given. After running the2-D gel, there are protocols for protein detection that include autoradiogra-

vi Preface

phy, and Coomassie, silver, and reversible metal chelate stains. 2-D ProteomeAnalysis Protocols covers image acquisition of the 2-D gel and has a detailedprotocol for computer analysis of the 2-D gel image. With the growing im-portance of the Internet, this book also includes protocols that enable readersto compare their results with other 2-D databases over the Internet or to con-struct their own 2-D resolved proteins, focusing on the latest mass spectrom-etry methods including MALDI-TOF-based peptide mapping, automated tandemmass spectrometry, and nanospray electrospray ionization technology.

Each chapter opens with a description of the protocol and the basic theorybehind the method. The Materials section lists all the equipment and reagentsrequired for carrying out the protocol. In the Methods section, each step ofthe protocol is listed in sufficient detail to successfully execute the experi-ment. Finally, the Notes section provides invaluable hints and alternatives fordealing with any problems or difficulties that may occur with the protocol.

Many people have contributed time and energy to make 2-D ProteomeAnalysis Protocols possible. A special thanks goes to all the contributors,each of whom made many important suggestions and improvements. I wishespecially to thank Jenny Fichmann, Reudi Aebersold, Thierry Rabilloud, MaryLopez, Wayne Patton, Lou Ramagli, Jean-Charles Sanchez, Marc Wilkins,Ron Appel, Angelike Görg, Vitaliano Pallini, and Denis Hochstrasser for in-valuable advice and help in preparing this book.

Andrew J. Link

vii

Contents

Preface ............................................................................................................ v

Contributors ................................................................................................... xiii

1 2-D Protein Gel Electrophoresis: An OverviewJenny Fichmann and Reiner Westermeier ....................................... 1

2 Solubilization of Proteins in 2-D Electrophoresis: An OutlineThierry Rabilloud ................................................................................ 9

3 Preparation of Escherichia coli Samples for 2-D Gel AnalysisRuth A. VanBogelen and Frederick C. Neidhardt .......................... 21

4 Preparing 2-D Protein Extracts from YeastDavid M. Schieltz ............................................................................... 31

5 2-D Protein Extracts from Drosophila melanogasterChrister Ericsson ............................................................................... 35

6 Preparing 2-D Protein Extracts from Caenorhabditis elegansRobert Zwilling ................................................................................... 43

7 Eukaryotic Cell Labeling and Preparation for 2-DNick Bizios .......................................................................................... 49

8 Differential Detergent Fractionation of Eukaryotic Cells: Analysis byTwo-Dimensional Gel Electrophoresis

Melinda L. Ramsby and Gregory S. Makowski ............................. 53

9 Fractionated Extraction of Total Tissue Proteins from Mouse andHuman for 2-D Electrophoresis

Joachim Klose ................................................................................... 67

10 Preparation and Solubilization of Body Fluids for 2-DJean-Charles Sanchez and Denis F. Hochstrasser ....................... 87

11 2-D Electrophoresis of Plant ProteinsAkira Tsugita and Masaharu Kamo ................................................. 95

12 Quantifying Protein in 2-D PAGE Solubilization BuffersLouis S. Ramagli ................................................................................ 99

13 Measuring the Radioactivity of 2-D Protein ExtractsAndrew J. Link and Nick Bizios ..................................................... 105

14 Advantages of Carrier Ampholyte IEFMary F. Lopez .................................................................................. 109

viii Contents

15 2-D Electrophoresis Using Carrier Ampholytes in the FirstDimension (IEF)

Mary F. Lopez .................................................................................. 111

16 Nonequilibrium pH Gel Electrophoresis (NEPHGE)Mary F. Lopez .................................................................................. 129

17 High-Resolution, 2-D Protein Electrophoresis Using NondedicatedEquipment

Marion Sarmiento ............................................................................. 133

18 Large-Gel 2-D ElectrophoresisJoachim Klose .................................................................................. 147

19 Advantages of Immobilized pH GradientsJenny Fichmann ............................................................................... 173

20 Casting Immobilized pH Gradients (IPGs)Elisabetta Gianazza ......................................................................... 175

21 Analytical IPG-DaltAngelika Görg and Walter Weiss ................................................... 189

22 IPG-Dalt of Very Alkaline ProteinsAngelika Görg .................................................................................. 197

23 Running Preparative Carrier Ampholyte and Immobilized pHGradient IEF Gels for 2-D

Neil M. Matsui, Diana M. Smith-Beckerman, Jenny Fichmann,and Lois B. Epstein .................................................................... 211

24 In-Gel Sample Rehydration of Immobilized pH GradientJean-Charles Sanchez, Denis Hochstrasser,

and Thierry Rabilloud ................................................................. 221

24A High-Resolution, IPG-Based, Mini Two-DimensionalGel Electrophoresis

Jean-Charles Sanchez and Denis F. Hochstrasser ..................... 227

25 Horizontal SDS-PAGE for IPG-DaltAngelika Görg and Walter Weiss ................................................... 235

26 Casting and Running Vertical Slab-Gel Electrophoresisfor 2D-PAGE

Bradley J. Walsh and Benjamin R. Herbert .................................. 245

27 Nonreducing 2-D Polyacrylamide Gel ElectrophoresisHong Ji and Richard J. Simpson ................................................... 255

28 2-D Diagonal Gel ElectrophoresisJoan Goverman ................................................................................ 265

Contents ix

29 2-D Phosphopeptide MappingHikaru Nagahara, Robert R. Latek, Sergei A. Ezhevsky,

and Steven F. Dowdy .................................................................. 271

30 Internal Standards for 2-DAndrew J. Link ................................................................................. 281

31 Autoradiography of 2-D GelsAndrew J. Link ................................................................................. 285

32 Double-Label AnalysisKelvin H. Lee and Michael G. Harrington ..................................... 291

33 Silver Staining of 2-D Electrophoresis GelsThierry Rabilloud ............................................................................. 297

34 Staining of Preparative 2-D Gels: Coomassie Blue andImidazole-Zinc Negative Staining

Neil M. Matsui, Diana M. Smith-Beckerman,and Lois B. Epstein ..................................................................... 307

35 Electroblotting of Proteins from 2-D Polyacrylamide GelsMichael J. Dunn ................................................................................ 313

36 Detection of Total Proteins on Western Blots of2-D Polyacrylamide Gels

Michael J. Dunn ................................................................................ 319

37 Protein Detection Using Reversible Metal Chelate StainsWayne F. Patton, Mark J. Lim, and David Shepro ....................... 331

38 Gylcoprotein Detection of 2-D Separated ProteinsNicolle H. Packer, Malcolm S. Ball, and Peter L. Devine ............ 341

39 Image Acquisition in 2-D ElectrophoresisWayne F. Patton, Mark J. Lim, and David Shepro ....................... 353

40 Computer Analysis of 2-D ImagesRon D. Appel and Denis F. Hochstrasser ..................................... 363

41 2-D Databases on the World Wide WebRon D. Appel, Amos Bairoch, and Denis F. Hochstrasser ......... 383

42 Comparing 2-D Electrophoresis Gels Across Internet DatabasesPeter F. Lemkin ................................................................................ 393

43 Constructing a 2-D Database for the World Wide WebRon D. Appel, Christine Hoogland, Amos Bairoch,

and Denis F. Hochstrasser ......................................................... 411

44 Absolute Quantitation of 2-D Protein SpotsSteven P. Gygi and Ruedi Aebersold ............................................ 417

45 Generating a Bacterial Genome Inventory:Identifying 2-D Spotsby Comigrating Products of the Genome on 2-D Gels

Ruth A. VanBogelen ........................................................................ 423

46 Immunoaffinity Identification of 2-DE Separated ProteinsBarbara Magi, Luca Bini, Barbara Marzocchi,

Sabrina Liberatoni, Roberto Raggiaschi,and Vitaliano Pallini .................................................................... 431

47 2-DE Spot Amino Acid Analysis with 9-FluroenylmethylChloroformate

Marc R. Wilkins, Jun X. Yan, and Andrew A. Gooley .................. 445

48 N-Terminal Amino Acid Sequencing of 2-DE SpotsMasaharu Kamo and Akira Tsugita ............................................... 461

49 Characterizing Proteins from 2-DE Gels by InternalSequence Analysis of Peptide Fragments:Strategies for Microsample Handling

Hediye Erdjument-Bromage, Mary Lui, Lynne Lacomis,and Paul Tempst .......................................................................... 467

50 Obtaining Molecular Weights of Proteins and Their CleavageProducts by Directly Combining Gel Electrophoresiswith Mass Spectrometry

Rachel R. Ogorzalek Loo, Joseph A. Loo,and Philip C. Andrews ................................................................ 473

51 Identification of Proteins by Matrix-Assisted LaserDesorption/Ionization Mass Spectrometry Using Peptideand Fragment Ion Masses

Paul L. Courchesne and Scott D. Patterson ................................. 487

52 Sample Preparation Methods for Mass Spectrometric PeptideMapping Directly from 2-DE Gels

Ole Nørregaard Jensen, Matthias Wilm, Andrej Shevchenko,and Matthias Mann ...................................................................... 513

53 Protein Identification and Analysis Tools in the ExPASy ServerMarc R. Wilkins, Elisabeth Gasteiger, Amos Bairoch,

Jean-Charles Sanchez, Keith L. Williams, Ron D. Appel,and Denis F. Hochstrasser ......................................................... 531

54 Automated Protein Identification Using Microcolumn LiquidChromatography-Tandem Mass Spectrometry

John R. Yates, III, Edwin Carmack, Lara Hays, Andrew J. Link,and Jimmy K. Eng ....................................................................... 553

x Contents

55 Peptide Sequencing of 2-DE Gel-Isolated Proteins byNanoelectrospray Tandem Mass Spectrometry

Ole Nørregaard Jensen, Matthias Wilm, Andrej Shevchenko,and Matthias Mann ...................................................................... 571

Index ............................................................................................................ 589

Contents xi

Contributors

RUEDI AEBERSOLD l Department of Molecular Biotechnology, Untversity of Washington, Seattle, WA

PHILLIP ANDREWS l University of Michigan Medical School, Ann Arbor, MI RON D. APPEL l Medical Informatics Division, Geneva University Hospital,

Geneva, Switzerland AMOS BAIROCH l Medical Biochemtstry Department, Untversity of Geneva,

Geneva, Switzerland MALCOLM S. BALL l MUCAB, Macquarie University, Sydney, Australta LUCA BINI l Department of Molecular Biology University of Siena, Siena,

Italy NICK BIZIOS l AGI Dermattcs, Freeport, NY EDWIN CARMACK l Department of Molecular Biotechnology, University of

Washtngton, Seattle, WA PAUL L. COURCHESNE 9 Protein Structure, Amgen, Inc., Thousand Oaks, CA PETER L. DEVINE l MUCAB, Macquarte University, Sydney, Austraba STEVEN F. DOWDY l Department of Pathology, Howard Hughes Medical

Institute, Washington University School of Medicine, St. Louis, MO MICHAEL J. DUNN l Department of Cardiothoracic Surgery, Nattonal Heart

and Lung Institute, Heart Science Centre, Harefteld Hospttal, Harefield, Middlesex, UK

JIMMY K. ENG l Department of Molecular Btotechnology, Untversity of Washtngton, Seattle, WA

LOIS B. EPSTEIN . Cancer Research Institute and the Department of Pediatrics, Untverstty of California, San Francisco, CA

HEDIYE ERDJUMENT-BROMAGE l Molecular Biology Program, Memorial Sloan- Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, NY

CHRISTER ERICSSON l Department of Biochemical Pharmacology, State University of New York at Buffalo, Buffalo, NY

SERGEI A. EZHEVSKY l Department of Pathology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louts, MO

JENNY FICHMANN l Amersham Pharmacia Biotech, San Francisco, CA

xiv Contributors

ELISABETH GASTEIGER l Medical Biochemistry Department, University of Geneva, Geneva, Switzerland

ELISABETTA GIANAZZA l Istituto di Scienze Farmacologiche, Universita di Milano, Milano, Italy

ANDREW A. GOOLEY l Macquarie University Centre for Analytical Biotechnology, The School of Biology and Chemistry, Macquarie Unt- versity, Sydney, Australia

ANGELIKA GORG 9 Lehrstuhl fur Allgemeine Lebensmitteltechnologie, Technische Universitiit Munchen, Freistng- Weihenstephan, Germany

JOAN M. GOVERMAN l Department of Molecular Biotechnology, University of Washington, Seattle, WA

STEVE P. GYGI . Department of Molecular Biotechnology, University of Washington, Seattle, WA

MICHAEL G. HARRINGTON l Department of Biology, California Institute of Technology, Pasadena, CA

LARA HAYS 9 Department of Molecular Btotechnology, University of Washington, Seattle, WA

BENJAMIN R. HERBERT . APAF, School of Biological Sciences, Macquarte University, Sydney, Australia

DENIS F. HOCHSTRASSER 9 Clinical Chemistry Laboratory, Medical Biochemistry Department, Geneva University Hospital, Geneva, Swit- zerland

CHRISTINE HOOGLAND 9 Medical Biochemistry Department, University of Geneva, Geneva, Switzerland

OLE NP~RREGAARD JENSEN l Protein Research Group, Department of Molecular Biology, Odense University, Odense, Denmark

HONG JI l Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia

MASAHARU KAMO l Research Institute for Biosciences, Science University of Tokyo, Chiba, Japan

JOACHIM KLOSE l Institut fur Humangenetik, Forschungshaus, Vtrchow- Klinikum, Berlin, Germany

LYNNE LACOMIS 9 Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, NY

ROBERT R. LATEK l Department of Pathology, Howard Hughes Medical Insti- tute, Washington University School of Medicine, St. Louis, MO

KELVIN H. LEE l Department of Chemical Engineering, Cornell University, Ithaca, NY

Contributors xv

PETER LEMKIN l Image Processing Section, Laboratory for Mathematical Biology, DBS, National Cancer Institute, FCRDC, Frederick, MD

SABRINA LIBERATORI l Department of Molecular Biology, University of Siena, Siena, Italy

MARK J. LIM l Microvascular Research Division, Biological Sciences Center, Boston University, Boston, MA

ANDREW J. LINK l Department of Molecular Biotechnology, University of Washington, Seattle, WA

MARY LUI l Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, NY

JOSEPH A. Loo 9 University of Michigan Medical School, Ann Arbor, MI RACHEL R. OGORZALEK Loo b University of Michigan Medical School, Ann

Arbor, MI MARV F. LOPEZ . ESA, Inc., Chelmsford, MA BARBARA MAGI l Department of Molecular Biology, University of Stena, Siena, Italy GREGORY S. MAKOWSKI l Department of Laboratory Medicine, Untverstty of

Connecticut Health Center, Farmington, CT MATTHIAS MANN l Protein and Peptide Group, European Molecular Biology

Laboratory, Heidelberg, Germany BARBARA MARZOCCHI l Department of Molecular Btology, University of Siena,

Siena, Italy NEIL M. MATSUI l Department of Hematology SFGH, San Francisco, CA HIKARU NAGAHARA l Department of Pathology, Howard Hughes Medical

Institute, Washington University School of Medtcine, St. Louis, MO FREDERICK C. NEIDHARDT 9 Department of Microbiology and Immunology, The

University of Michigan Medical School, Ann Arbor, MI NICOLLE H. PACKER 9 MUCAB, Macquarie University, Sydney, Australia V~TALIANO PALLINI l Department of Molecular Biology, Section of Biological

Chemistry, University of Siena, Siena, Italy SCOTT D. PATTERSON l Protein Structure, Amgen, Inc., Thousand Oaks, CA WAYNE F. PATTON 9 Bioanalytical Assay Development and Validation,

Molecular Probes, Inc , Eugene, OR THIERRY RABILLOUD l CEA-Laboratorie de Bioenergbtique Cellulaire et

Pathologique- UA 2019, DBMS/BMCC, Grenoble, France ROBERTO RAGGIASCHI l Department of Molecular Biology, University of Siena,

Siena, Italy LOUIS S. RAMAGLI l Department of Molecular Genetics, M. D. Anderson

Cancer Center, Houston, TX

xvi Contributors

MELINDA L. RAMSBY l School of Medicine, University of Connecticut Health Center, Farmington, CT

JEAN-CHARLES SANCHEZ l Clintcal Chemistry Laboratory, Geneva University Hospttal, Geneva, Switzerland

MARION SARMIENTO l Hospital Director, Veterinary Diagnostics, Houston, TX DAVID M. SCHIELTZ 9 Department of Molecular Biotechnology, University of

Washington, Seattle, WA DAVID SHEPRO l Microvascular Research Diviston, Btological Sciences Cen-

ter, Boston University, Boston, MA ANDREJ SHEVCHENKO l Protein and Pepttde Group, European Molecular

Biology Laboratory, Heidelberg, Germany RICHARD J. SIMPSON 9 Ludwig Instttute for Cancer Research, Royal Melbourne

Hospital, Parkville, Victoria, Australia DIANA M. SMITH-BECKERMAN 9 Centerfor Biomedical Science, San Francisco State

Universtty, San Francisco, CA PAUL TEMPST l Molecular Biology Program, Memorial Sloan-Kettering

Cancer Center and Cornell University Graduate School of Medical Sciences, New York, NY

AKIRA TSUGITA l Research Institute for Biosciences, Sctence University of Tokyo, Chiba, Japan

RUTH A. VANBOGELEN l Biotechnology Laboratory, Parke-Davis Pharmaceutical Research, Ann Arbor, MI

BRADLEY J. WALSH l APAF, School of Biological Sciences, Macquarie Untversity, Sydney, Australia

WALTER WEISS b Lehrstuhlfiir Allgemeine Lebensmitteltechnologie, Technische Universitat Miinchen, Freising- Weihenstephan, Germany

REINER WESTERMEIER l ETC Electrophorese-Technik, Westermeier & Schickle GmbH, Germany

MARC R. WILKINS l Macquarie University Centre for Analyttcal Biotechnology, School of Btological Sctences, Macquarie Universtty, Sydney, Australia

KEITH L. WILLIAMS l Australian Proteome Analysis Facility, Macquarie University, Sydney, Australia

MATTHIAS W~LM l Protein and Peptide Group, European Molecular Biology Laboratory, Heidelberg, Germany

JUN X. YAN l Macquarie University Centre for Analytical Biotechnology, The School of Biology and Chemistry, Macquarie University, Sydney, Australia

Contributors xvii

JOHN R. YATES, III l Department of Molecular Biotechnology, Universzty of Washington, Seattle, WA

ROBERT ZWJLLING l Fakulttitftir Biologie, Universittit Heidelburg, Heidelburg, Germany