HPLC of Peptides and Proteins [Methods In Molec Bio, Vol 251] - M. Aguilar (Humana, 2004) WW.pdf

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METHODS IN MOLECULAR BIOLOGY TM
Volume 251
HPLC of Peptides
Methods and Protocols
Edited by
Marie-Isabel Aguilar
METHODS IN MOLECULAR BIOLOGY TM
HPLC of Peptides
and Proteins
and Proteins
Methods and Protocols
Edited by
Marie-Isabel Aguilar
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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
HPLC of Peptides
and Proteins
Methods and Protocols
Edited by
Marie-Isabel Aguilar
Department of Biochemistry and Molecular Biology
Monash University, Clayton, Victoria, Australia
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© 2004 Humana Press Inc.
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All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in
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without written permission from the Publisher. Methods in Molecular Biology TM is a trademark of The
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All authored papers, comments, opinions, conclusions, or recommendatoins are those of the author(s), and
do not necessarily reflect the views of the publisher.
This publication is printed on acid-free paper. ∞
ANSI Z39.48-1984 (American Standards Institute Permanence of Paper for Printed Library Materials.)
Cover design by Patricia F. Cleary.
Cover illustration: Cartoon depicting the selective retention of a peptide from a mixture of peptides and
proteins. Illustration by Lee Tzong-Hsien, William Farrugia, and Marie-Isabel Aguilar.
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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1
ISSN: 1064-3745
E-ISBN: 1-59259-742-4
Library of Congress Cataloging-in-Publication Data
HPLC of peptides and proteins : methods and protocols / edited by
Marie-Isabel Aguilar.
p. ; cm. -- (Methods in molecular biology ; 251)
Includes bibliographical references and index.
ISBN 0-89603-977-3 (alk. paper)
1. Peptides--Analysis. 2. Proteins--Analysis. 3. High performance liquid chromatography.
[DNLM: 1. Peptides--chemistry. 2. Chromatography, High Pressure Liquid--methods. 3. Proteins--
chemistry. QU 68 H872 2004] I. Aguilar, Marie-Isabel. II. Methods in molecular biology (Totowa, N.J.);
v. 251.
QP552.P4H73 2004
572'.6536--dc22
200301746 4
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Preface
The introduction of high-performance liquid chromatography (HPLC)
to the analysis of peptides and proteins some 25 years ago revolutionized the
biological sciences by enabling the rapid and sensitive analysis of peptide and
protein structure through the exquisite speed, sensitivity, and resolution that
can be easily obtained. Today, HPLC in its various modes has become the
pivotal technique in the characterization of peptides and proteins and currently
plays a critical role in both our understanding of biological processes and in
the development of peptide- and protein-based pharmaceuticals.
The number of applications of HPLC in peptide and protein purification
continues to expand at an extremely rapid rate. Solid-phase peptide synthesis
and recombinant DNA techniques have allowed the production of large
quantities of peptides and proteins that need to be highly purified. HPLC
techniques are also used extensively in the isolation and characterization of
novel proteins that will become increasingly important in the postgenomic
age. The design of multidimensional purification schemes to achieve high levels
of product purity further demonstrates the power of HPLC techniques not only
in the characterization of cellular events, but also in the production of peptide-
and protein-based therapeutics. HPLC continues to be at the heart of the
analytical techniques with which scientists in both academia and in industry
must arm themselves to be able to fully characterize the identity, purity, and
potency of peptides and proteins.
The aim of HPLC of Peptides and Proteins: Methods and Protocols is to
provide the beginner with a sufficiency of the practical information needed to
develop separation and analytical protocols for peptide and protein analysis.
This volume opens with an overview of the basic theory and general
methodology of HPLC, with particular reference to the key separation
parameters that can be manipulated to achieve high resolution. Each of the
commonly used HPLC techniques are covered in Chapters 2–9, whereas
methods for capillary to large-scale preparative isolation are described in
Chapters 10–15. Chapters 16–27 provide those already experienced in HPLC
with a number of specific applications, as in case studies to illustrate the
analytical approaches to a particular separation or assay challenge, with
examples drawn from contemporary fields in biochemistry and biotechnology.
These applications include proteolytic mapping, posttranslational
modifications, neuropeptide processing, glycopeptides and glycoproteins,
v
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vi
Preface
MHC-binding peptides, toxins/venoms, membrane proteins, antibodies,
combinatorial and proteome analysis, and enzymatic activity.
HPLC of Peptides and Proteins: Methods and Protocols will be a valuable
resource for a wide range of scientists, including biochemists, molecular
biologists, pharmacologists, and microbiologists, who work with peptides and/
or proteins in both academic and biotechnology laboratories.
Finally, I would like to thank all of the authors for their enthusiastic
participation and excellent contributions.
Marie-Isabel Aguilar
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