Table Of ContentSequence Analysis:
Current
Methodologies
Editor
Ajit S. Bhown, Ph.D.
Associate Professor
Division of Clinical Immunology and Rheumatology
Department of Medicine
University of Alabama at Birmingham
Birmingham, Alabama
Boca Raton London New York
CRC Press, Inc.
CRC Press is an imprint of the
Boca Raton, Florida
Taylor & Francis Group, an informa business
Boca Raton London New York
CRC Press is an imprint of the
Taylor & Francis Group, an informa business
First published 1988 by CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742
Reissued 2018 by CRC Press
© 1988 by CRC Press, Inc.
CRC Press is an imprint of Taylor & Francis Group, an Informa business
No claim to original U.S. Government works
This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish
reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the
consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this
publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has
not been acknowledged please write and let us know so we may rectify in any future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form
by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording,
or in any information storage or retrieval system, without written permission from the publishers.
For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.
com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a
not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a
photocopy license by the CCC, a separate system of payment has been arranged.
Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and
explanation without intent to infringe.
Library of Congress Cataloging-in-Publication Data
Protein/peptide sequence analysis.
Bibliography: p.
Includes index.
1. Amino acid sequence. I. Bhown, Ajit S..
1934-
QP551.P69745 1987 574.19’245 87-718
ISBN 0-8493-6583-X
A Library of Congress record exists under LC control number: 87000718
Publisher’s Note
The publisher has gone to great lengths to ensure the quality of this reprint but points out that some imperfections in the original copies
may be apparent.
Disclaimer
The publisher has made every effort to trace copyright holders and welcomes correspondence from those they have been unable to
contact.
ISBN 13: 978-1-315-89698-4 (hbk)
ISBN 13: 978-1-351-07608-1 (ebk)
Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the
CRC Press Web site at http://www.crcpress.com
PREFACE
The functional properties of a protein nlolecule have long been known to depend upon
its primary structure. It is, therefore, knowledge of the amino acid sequence that allows an
investigator to understand: the interaction between an antibody molecule and its antigen; the
ability of a hormone to bind to a receptor; the ability of an enzyme to bind and cleave its
substrate with the ability for a variety of molecules to form aggregates and, hence, develop
a structural framework upon which other biological activities occur. The fascination of protein
chemistry, therefore, depends very much upon our ability to gain information regarding the
primary structure of protein molecules and how that amino sequence directs the molecular
function. Even with the new excitement generated by the very rapid methods allowing DNA
sequencing to be transformed into protein structure, we still have an enormous need to
determine amino acid sequences on ever-decreasing amounts of available polypeptide sam-
ples. Since structure and function are so intimately involved in proteins, even our knowledge
of the DNA sequence does not always allow us to make a leap to functional relevance
without first knowing and determining at least some parts of the amino acid sequence of a
given protein molecule. This book is an attempt to provide in a single source current state-
of-the-art methodologies for protein sequence analysis. It is hoped that these various chapters
are presented in such a way that both the newcomer and the established protein chemist will
find useful information and directions to new techniques.
The contributors to this volume have been carefully selected by the editor from among
the world leaders in this field. Each chapter is written in a way that establishes the individual
author's particular perspective on the field of his research. Chapter 1 outlines experimental
details for the purification of proteins and peptides suitable for sequence analysis by high
pressure liquid chromatography. Chapters 2 through 5 describe in detail how the purified
protein-peptide can be subjected to sequence analysis. Use of homologous coupling reagents
has been described in Chapter 6, while amino acid sequence analysis from the carboxyter-
mina1 end by chemical and enzymatic procedures has been discussed in Chapters 7 and 8,
respectively. Mass spectrometry as a tool for sequence analysis is discussed in Chapter 9,
and Chapter 10 covers the novel methods of amino acid sequence analysis. The last two
chapters (1 1 and 12) deal with the identification procedures of modified (methylated) and
derivatized (phenylthiohydantoin) amino acids.
This book offers a rich array of techniques and methods for sequencing proteins and
peptides. It should meet the expectations of investigators in protein chemistry who wish to
update their knowledge of sequencing techniques, and of those who wish to reacquaint
themselves with the best available current technologies.
J. Claude Bennett
THE EDITOR
Ajit S. Bhown, Ph.D. is a Research Associate Professor in the Division of Clinical
Immunology and Rheumatology, Department of Medicine, the University of Alabama at
Birmingham, Birmingham, Alabama where he heads the Protein Sequencing Core Facility.
Dr. Bhown obtained his Ph.D. degree in 1965 from the University of Rajasthan, Jaipur,
India, and his post-doctoral training at the University of Tennessee Medical Center, Knox-
ville, from 1965 to 1967 and at the University of Alabama at Birmingham from 1974 to
1975. He was on the Teaching Faculty in the College of Medicine in Jodhpur, India from
1967 to 1974. His major research interest has been in the development of techniques for
the structural studies of proteins and peptides. Dr. Bhown has presented numerous lectures
at international and national scientific meetings and seminars and has published more than
80 scientific papers and chapters.
CONTRIBUTORS
J. Claude Bennett, M.D. James J. L'Italien, Ph.D.
Professor and Chairman Senior Scientist
Department of Medicine Molecular Genetics, Inc.
University of Alabama at Birmingham Minnetonka, Minnesota
Birmingham, Alabama
Woon Ki Paik, M.D.
Russell F. Doolittle, Ph.D.
Professor
Professor
Fels Research Institute
Department of Chemistry
Temple University School of Medicine
University of California, San Diego
Philadelphia, Pennsylvania
La Jolla, California
Minnie Rangarajan, Ph.D.
Rikimaru Hayashi, Ph.D.
Research Officer
Associate Professor
Centre for Biotechnology
Research Institute for Food Science
Department of Biochemistry
University of Kyoto
Imperial College
Uji, Kyoto, Japan
London, England
Michael W. Hunkapiller, Ph.D.
Johann Salnikow, Ph.D.
Scientific Director
Professor
Applied Biosystems
Institute of Biochemistry and Molecular
Foster City, California
Biology
Technical University Berlin
Rodney A. Jue, Ph.D.
Berlin, West Germany
Senior Research Scientist
Immunology Department
Jin Shan-Wei
Hybritech, Inc.
Senior Scientist
San Diego, California
Shanghai Institute of Organic Chemistry
Shanghai, China
Sangduk Kim, Ph.D.
Associate Professor
Fels Research Institute George E. Tarr, Ph.D.
Temple University School of Medicine Lecturer
Philadelphia, Pennsylvania Department of Biochemistry
University of Michigan
Henry C. Krutzsch, Ph.D. Ann Arbor, Michigan
Cancer Expert
Laboratory of Pathology Kenneth J. Wilson, Ph.D.
National Cancer Institute Research and Development Scientist
National Institutes of Health Applied Biosystems, Inc.
Bethesda, Maryland Foster City, California
Hyang Woo Lee, Ph.D. B. Wittmann-Liebold, Ph.D.
Associate Professor Professor
Department of Biochemistry Molecular Genetics
Sung Kyun Kwan University Max Planck Institute
Suwon, Kyunggi-do, Korea Berlin, West Germany
ACKNOWLEDGMENTS
I would like to express my sincere gratitude to the authors who have helped me in producing
this book. I appreciate the support given to me by Dr. J. Claude Bennett and his help in
the completion of this book. I am grateful for the critique of the Advisory Board which was
helpful in the selection of the authors and subject matter. I would like to thank Ms. Sandra
Reid for the typing responsibilities. I am also grateful to CRC Press for publishing this
book.
In memory of my Parents
This book is aflectionately dedicated to my wife Meera, daughter Sonchita,
and son Abhoyjit.
TABLE OF CONTENTS
Chapter 1
Purification of ProteinsJPeptides for Structural Studies ...................................1
K. J. Wilson
Chapter 2
Manual Methods for ProteinJPeptide Sequence Analysis ................................3 5
G. E. Tarr
Chapter 3
Liquid Phase Sequence Analysis of ProteinsJPeptides ................................... 49
A. S. Bhown and J. C. Bennett
Chapter 4
Solid-Phase Sequence Analysis of ProteinsIPeptides.. ..................................7.3
J. J. L'Italien
Chapter 5
Gas Phase Sequence Analysis of ProteinsIPeptides ......................................8 7
M. W. Hunkapiller
Chapter 6
Homologous Edman-Type Reagents in Microsequence Analysis of Polypeptides ...... .l19
B. Wittman-Liebold, J. Shan-Wei, and J. Salnikow
Chapter 7
Chemical Methods of Amino Acid Sequence Analysis from Carboxyterminal End .... 135
M. Rangarajan
Chapter 8
Enzymatic Methods of ProteidPeptide Sequence from Carboxyterminal End ..........1 45
R. Hayashi
Chapter 9
ProteidPeptide Sequence Analysis by Mass Spectrometry.. ...........................,1 61
H. C. Krutzsch
Chapter 10
A Novel Approach to Amino Acid Sequencing .......................................1.8 1
R. A. Jue and R. F. Doolittle
Chapter 11
Analysis of Modified Amino Acids ...................................................1.9 3
W. K. Paik, H. W. Lee, and S. Kim
Chapter 12
Identification of Phenylthiohydantoin Derivatives of Amino Acids.. ................... 211
A. S. Bhown and J. C. Bennett
