Peptide Analysis Protocols

Methods in Molecular Biology John M. Walker, SERIES EDITOR

36. Pep t ide Analysis Protocols , edited by Ben M. Dunn and Michael W. Pennington, 1994

35. Pep t ide Synthesis Protocols , edited by Michael W. Pennington and Ben M. Dunn, 1994

34. Immunocytochemica l Methods and Protocols , edited by Lorette C. Javois, 1994

33. In Si tu Hybrid iza t ion Protocols , edited by K. H. Andy Choo, 1994 32. Basic P ro te in and Pep t ide Protocols, edited by John M. Walker, 1994 31. Protocols for Gene Analysis, edited by Adrian J. Harwood, 1994 30. DNA-Prote in Interactions, edited by G. GeoffKneale, 1994 29. Chromosome Analysis Protocols, edited by John R. Gosden, 1994 28. Protocols for Nucleic Acid Analysis by Nonradioact ive Probes, edited by

Peter G. Isaac, 1994 27. B iomembrane Protocols: II. Architecture and Function, edited by

John M. Graham and Joan A. Higgins, 1994 26. Protocols for OHgonucleotide Conjugates, edited by Sudhir Agrawal, 199~ 25. Computer Analysis of Sequence Data: Part II, edited by

Annette M. Griffin and Hugh G. Griffin, 1994 24. Computer Analysis of Sequence Data: Part I, edited by

Annette M. Griffin and Hugh G. Griffin, 1994 23. DNA Sequencing Protocols, edited by Hugh G. Griffin

and Annette M. Griffin, 1993 22. Optical Spectroscopy, Microscopy, and Macroscopic Techniques,

edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomas, 1994

21. Protocols in Molecular Parasitology, edited by John E. Hyde, 1993 20. Protocols for Oligonucleotides and Analogs, edited by

Sudhir Agrawal, 1993 19. B iomembrane Protocols: I. Isolation and Analysis, edited by

John M. Graham and Joan A. Higgins, 1993 18. Transgenesis Techniques, edited by David Murphy

and David A. Carter, 1993 17. Spectroscopic Methods and Analyses, edited by Christopher Jones,

Barbara Mulloy, and Adrian H. Thomas, 1993 16. Enzymes of Molecular Biology, edited by Michael M. Burrell, 1993 15. PCR Protocols, edited by Bruce A. White, 1993 14. Glycoprotein Analysis in Biomedicine, edited by Elizabeth F. Hounsell,

1993 13. Protocols in Molecular Neurobiology, edited by Alan Longstaf f

and Patricia Revest, 1992 12. Pulsed-Field Gel Electrophoresis , edited by Margit Burmeister

and Levy Ulanovsky, 1992 11. Prac t ica l Pro te in Chromatography, edited by Andrew Kenney

and Susan Fowell, 1992 10. Immunochemica l Protocols , edited by Margaret M. Manson, 1992 9. Protocols in Human Molecular Genetics, edited by

Christopher G. Mathew, 1991

Earlier volumes are stil l available, Contact Humana for details,

Peptide Analysis Protocols

Edited by

Ben M. D u n n University of Florida, Gainesville, FL

and

M i c h a e l W. P e n n i n g t o n Bachem Bioscience, King of Prussia, PA

Humana Press ~ Totowa, New Jersey

© 1994 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

All rights reserved.

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permis- sion from the Publisher.

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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1

Library of Congress Cataloging in Publication Data

Peptide analysis protocols/edited by Ben M. Dunn and Michael W. Pennington p. cm.--(Methods in molecular biology; 36)

Companion v. to: Peptide synthesis protocols. Includes index. ISBN 0-89603-274-4 (acid-free paper) 1. Peptides--Analysis--Laboratory manuals. I. Dunn, Ben M. II. Pennington, Michael W.

III. Peptide synthesis protiocols. IV. Series: Methods in molecular biology (Totowa, N.J.); 36 QP552.P4P43 1994 574.19' 2456--dc20 94-22663

CIP

Preface

As the technology base for the preparation of increasingly com- plex peptides has improved, the methods for their purification and analy- sis have also been improved and supplemented. Peptide science routinely utilizes tools and techniques that are common to organic chemistry, pro- tein chemistry, biophysical chemistry, enzymology, pharmacology, and molecular biology. A fundamental understanding of each of these areas is essential for interpreting all of the data that a peptide scientist may see.

The purpose of Peptide Analysis Protocols is to provide the novice with sufficient practical information necessary to begin developing useful analysis and separation skills. Understanding and developing these skills will ultimately yield a scientist with broadened knowledge and good problem-solving abilities. Although numerous books that address dif- ferent specialties, such as HPLC, FAB-MS, CE, and NMR, have been written, until now no single volume has reviewed all of these techniques with a focus on "getting started" in separation and analysis of peptides.

This volume will also provide those who already possess practical knowledge of the more advanced aspects of peptide science with detailed applications for each of these protocols. Because the chapters have been written by researchers active in each of the fields that they discuss, a great deal of information on and insight into solution of real problems that they have encountered is presented. Examplary results are clearly demonstrated and discussed. For more advanced investiga- tions, supplementary experiments are often suggested.

Additionally, Peptide Analysis Protocols contains several applica- tions and extensions of peptide science. Thus, we include chapters on such topics as the construction of peptide libraries, recombinant pep- tide synthesis, immunochemical methods used for peptides, the kinetic analysis of proteases using peptide substrates, and de novo protein design and synthesis. These represent state-of-the-art technologies and

vi Preface

applications that make this volume complete when used in combination with its companion work in this series, Peptide Synthesis Protocols.

It is hoped that after reading Peptide Analysis Protocols and applying the techniques described, the reader can develop successful separations, analyses, and applications of each of his or her peptides.

We wish to thank the authors for their excellent contributions and cooperation.

Ben M. Dunn Michael W. Pennington

C o n t e n t s

Preface ............................................................................................................................ v Companion Volume Contents ..................................................................................... ix

Contributors ................................................................................................................. xi

CH. 1.

CH. 2.

CH. 3.

CH. 4.

CH. 5.

CH. 6.

CH. 7.

CH. 8.

CH. 9.

CH. 10.

CH. 11.

Gel-Filtration Chromotography, Daniel M. Bollag ..................................................................................... 1

Ion-Exchange Chromatography, Danie l M. Bollag ................................................................................... 11

Reversed-Phase HPLC: Analytical Procedure, Udo Nirenberg ....................................................................................... 23

Reversed-Phase High-Performance Liquid Chromatography: A Semipreparative Methodology,

Michae l E. Byrnes ................................................................................. 37 Applications of Strong Cation-Exchange (SCX)-HPLC

in Synthetic Peptide Analysis, Dan L. Cr immins ................................................................................... 53

Principles and Practice of Peptide Analysis with Capillary Zone Electrophoresis,

Thomas E. Wheat .................................................................................. 65 Fast Atom Bombardment Mass Spectrometric Characterization

of Peptides, P. R. Das and B. N. Praman ik .............................................................. 85

Sequence Analysis of Peptide Resins from Boc/Benzyl Solid-Phase Synthesis,

Jan Pohl ............................................................................................... 107 NMR Spectroscopy of Peptides and Proteins,

M a r k G. Hinds and R a y m o n d S. Norton ........................................... 131 Techniques for Conjugation of Synthetic Peptides

to Carrier Molecules, J. M a r k Carter ..................................................................................... 155

Epitope Prediction Methods, J. M a r k Carter ..................................................................................... !93

vii

viii Contents

Crt. 12. Epitope Mapping of a Protein Using the Geysen (PEPSCAN) Procedure,

J. Mark Carter ..................................................................................... 207 CH. 13. Analysis of Proteinase Specificity by Studies of Peptide Substrates:

The Use of UV and Fluorescence Spectroscopy to Quantitate Rates of Enzymatic Cleavage,

Ben M. Dunn, Paula E. Scarborough, Ruth Davenport, and Wieslaw Swietnicki ............................................................. 225

Cia. 14. Synthesis of Recombinant Peptides, Gino Van Heeke, Jay S. Stout, and Fred W. Wagner ...................... 245

CH. 15. De Novo Design of Proteins: Template-Assembled Synthetic Proteins (TASP),

Gabriele Tuchscherer, Verena Steiner, Karl-Heinz Altmann, and Man fred Mutter .................................................................. 261

CH. 16. Chemical Synthesis of the Aspartic Proteinase from Human Immunodeficiency Virus (HIV),

Paul D. Hoeprich, Jr ........................................................................... 287 CH. 17. Multiple and Combinatorial Peptide Synthesis: Chemical Development

and Biological Applications, Philip C. Andrews, Daniele M. Leonard, Wayne L. Cody,

and Tomi K. Sawyer ................................................................... 305 Index .......................................................................................................................... 329

Contents of the companion volume:

Peptide Synthesis Protocols (Note: Cross references to these chapters will include the notation PSP)

Preface Companion Volume Contents Contributors

CH. 1.

CH. 2.

CH. 3.

CH. 4.

CH. 5.

CH. 6.

CH. 7.

CH. 8.

CH. 9.

CH. 10.

CH. 11.

Procedures to Improve Difficult Couplings, Michael W. Pennington and Michael E. Byrnes

Methods for Removing the Fmoc Group, Gregg B. Fields

Solvents for Solid-Phase Peptide Synthesis, Cynthia G. Fields and Gregg B. Fields

HF Cleavage and Deprotection Procedures for Peptides Synthesized Using a Boc/Bzl Strategy,

Michael W. Pennington Acid Cleavage/Deprotection in Fmoc/tBu Solid-Phase Peptide Synthesis,

Fritz Dick Bromoacetylated Synthetic Peptides: Starting Materials for Cyclic

Peptides, Peptomers, and Peptide Conjugates, Frank A. Robey

Formation of Disulfide Bonds in Synthetic Peptides and Proteins, David Andreau, Fernando Albericio, Ndria A. Sol~,

Mark C. Munson, Marc Ferrer, and George Barany Site-Specific Chemical Modification Procedures,

Michael W. Pennington Synthesis of Phosphopeptides Containing O-Phosphoserine

and O-Phosphothreonine, Anatol Arendt and Paul A. Hargrave

Solid-Phase Synthesis of Phosphorylated Tyr-Peptides by "Phosphite Triester" Phosphorylation,

Michael W. Pennington Design of Novel Synthetic Peptides Including Cyclic Conformationally

and Topographically Constrained Analogs, Victor J. Hruby and G. Gregg Bonner

ix

x Companion Volume Contents

Cn. 12.

Cml3.

CH. 14.

Cn. 15.

Solid-Phase Synthesis of Peptides Containing the CH2NH Reduced Bond Surrogate,

Michael W. Pennington Approaches to the Asymmetric Synthesis of Unusual Amino Acids,

Victor J. Hruby and Xinhua Qian Synthesis of Fully Protected Peptide Fragments,

Monika Mergler Peptide Synthesis via Fragment Condensation,

Rolf Nyfeler

Index

Contr ibutors

KARL-HEINZ ALTMANN • Section de Chimie, Universite de Lausanne, Lausanne, Switzerland. Present Address: Ciba-Geigy AG, Basel, Switzerland

PHILIP C . ANDREWS • Department of Biological Chemistry, University of Michigan, Ann Arbor, MI

DANIEL M. BOLLAG • Merck, Sharpe, and Dohme, West Point, PA MICHAEL E. BYRNES • Bachem Bioscience, King of Prussia, PA J. MARK CARTER • Cytogen, Princeton, NJ WAYNE L. CODY • Department of Chemistry, Parke-Davis

Pharmaceutical Research, Division of Warner-Lambert Co., Ann Arbor, MI

DAN L. CRIMMINS • Core Protein/Peptide Facility, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO

P. R. DAS • Schering-Plough Research Institute, Kenilworth, NJ RUTH DAVENPORT • Department of Biochemistry and Molecular Biology,

College of Medicine, University of Florida, Gainesville, FL BEN M. DONN • Department of Biochemistry and Molecular Biology,

College of Medicine, University of Florida, Gainesville, FL MARK G. HINDS • NMR Laboratory, Biomolecular Research Institute,

Parkville, Australia PAUL D. HOEPRICH, JR. • Applied Biosystems Inc., Foster City, CA DANIELE M . LEONARD • Department of Chemistry, Parke-Davis

Pharmaceutical Research, Warner-Lambert Co., Ann Arbor, MI MANFRED MUTTER • Section de Chimie, Universite de Lausanne,

Lausanne, Switzerland UDO NIRENBERG • Bachem Feinchemikalien AG, Bubendo~

Switzerland

xi

xii Contributors

RAYMOND S. NORTON • NMR Laboratory, Biomolecular Research Institute, Parkville, Australia

JAN POnE "Microchemical Facility, Winship Cancer Center, Emory University School of Medicine, Atlanta, GA

B. N. PRAMANIK • Schering-Plough Research Institute, Kenilworth, NJ TOMI K. SAWYER • Department of Chemistry, Parke-Davis

Pharmaceutical Reserach, Warner-Lambert Co., Ann Arbor, MI PAULA E. SCARBOROUGH • Department of Pathology, Duke University,

Durham, NC VERENA STEINER • Section de Chimie, Universite de Lausanne,

Lausanne, Switzerland. Present Address: Ciba-Geigy AG, Basel, Switzerland

JAY S. STOUT • BioNebraska Inc., Lincoln, NE WIESLAW SwIEr~Cra • Department of Biochemistry and Molecular

Biology, College of Medicine, University of Florida, Gainesville, FL GABRmLE TtJCHSCHERER • Section de Chimie, Universite de Lausanne,

Lausanne, Switzerland GINO VAN HEEKE • Protein Expression Laboratory, Interdisciplinary

Center for Biotechnology Research, University of Florida, Gainesville, FL; BioNebraska, Inc. Lincoln, NE

FRED W. WAGNER • BioNebraska Inc., Lincoln, NE; Department of Biochemistry, University of Nebraska, Lincoln, NE

THOMAS E. WHEAT • Analytical Technology Inc., Boston, MA