Posttranslational Modifications of Proteins: Tools for Functional Proteomics

336 Pages·2002·1.913 MB·English

by  Jiang Wu, J. Throck Watson (auth.), Christoph Kannicht (eds.)

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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 TM
Posttranslational
Modifications
of Proteins
Tools for Functional Proteomics
Edited by
Christoph Kannicht
Institut für Molekularbiologie und Biochemie,
FreieUniversität Berlin, Berlin, Germany
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Cover illustration: The figure shows the effect of a posttranslational modification (here N-glycosylation) on
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Library of Congress Cataloging in Publication Data
Posttranslational modifications of proteins : tools for functional proteomics / edited by
Christoph Kannicht.
p. cm. -- (Methods in molecular biology ; v. 194)
Includes bibliographical references and index.
ISBN 0-89603-678-2
1.Post-translational modification--Laboratory manuals. I. Kannicht, Christoph. II.
Series.
QH450.6 .P685 2002
572'.6--dc21
20010511864
Preface
Posttranslational Modifications of Proteins: Tools for Functional Proteomics is
a compilation of detailed protocols needed to detect and analyze the most important
co- and posttranslational modifications of proteins. Though, for reasons of simplicity
not explicitly mentioned in the title, both kinds of modifications are covered, whether
they occur during, or after, biosynthesis of the protein. My intention was to cover the
most significant protein modifications, focusing on the fields of protein function,
proteome research, and the characterization of pharmaceutical proteins.
The majority of all proteins undergo co- and/or posttranslational modifications.
Knowledge of these modifications is extremely important, since they may alter
physical and chemical properties, folding, conformation distribution, stability, activ-
ity, and, consequently, function of the proteins. Moreover, the modification itself can
act as an added functional group. Examples of the biological effects of protein modi-
fications include: phosphorylation for signal transduction, ubiquitination for pro-
teolysis, attachment of fatty acids for membrane anchoring or association,
glycosylation for protein half-life, targeting, cell–cell and cell–matrix interactions,
and carboxylation in protein–ligand binding to name just a few. Full understanding
of a specific protein structure–function relationship requires detailed information not
only on its amino acid sequence, which is determined by the corresponding DNA
sequence, but also on the presence and structure of protein modifications.
The individual chapters provide detailed step-by-step instructions for the analysis
of the most important protein modifications, especially the assignment of disulfide
bond sites in proteins (Chapter 1). Analysis of protein glycosylation is treated in great
detail. Starting with the analysis of carbohydrate composition (Chapter 2), the meth-
ods for cleavage, labeling, separation, and sequence analysis of N-linked glycans are
described (Chapters 3–5). Analysis of protein O-glycosylation in general and spe-
cific detection of O-linkedN-acetyglucosamine residues follow (Chapters 6 and 7,
respectively). Finally, Chapter 8 provides a method to analyze the oligosaccharides
present at specific single glycosylation sites in a protein.
A method for analysis of glycosylphosphatidylinositols describes analysis of
the carbohydrate and lipid portions as well (Chapter 10). Two more protocols on
the analysis of lipid modifications, in particular S-acylation (Chapter 11) and
ubiquitination (Chapter 12) follow.
Further protein modifications of interest and different nature complete the
book: analysis of protein methylation, acetylation, phosphorylation, and sulfation
v
vi Preface
(inChapters 13–15, respectively), and analysis of α-amidation,γ-glutamate, iso-
aspartate, and lysine hydroxylation in Chapters 16–19. Topics of more general
interest are treated in the final two chapters. Chapter 20 describes the use of a
heterologous expression system for the analysis of posttranslational modifica-
tions and Chapter 21 shows how to detect the influence of glycosylation on
protein spot patterns in 2D gel electrophoresis.
Let me give special mention to two areas of research of high current interest:
the fields of (1) proteomics and (2) the characterization of biological pharmaceu-
ticals. (1) With respect to proteomics, research in the field of genomics has led
to knowledge of the complete human DNA sequence. Measurement of the mRNA
pool at a specific status of the cell, the “transcriptome,” was found to not neces-
sarily reflect the cells’ actual protein expression pattern. In proteomics research,
the description of expression levels of proteins related to a defined cell or tissue
status will be incomplete without knowledge of the posttranslational modifica-
tions of those proteins.
In addition to possible changes in the activity or function of a protein, changes
in its molecular weight or charge caused by protein modifications will influence
the separation of proteins during 2D gel electrophoresis. Protein spot patterns
generated by 2D electrophoresis will change because of altered protein expres-
sion or changes in the protein modifications. As an example, Chapter 21 describes
detection and influence of sialylation and N-glycosylation on the protein spot
pattern obtained by 2D gel electrophoresis.
(2) An additional important practical application of posttranslational modifica-
tion analysis is to ensure the product quality of therapeutic pharmaceutical pro-
teins. Recombinant proteins intended for therapeutic use in humans must be
accorded particularly thorough investigation. Product quality depends on accurate
posttranslational modification in the respective expression system during produc-
tion, e.g., insect or several mammal cell lines. Note that different expression sys-
tems may vary in their ability to carry out posttranslational modifications and that
changes in cell culture conditions also influence these modifications. Thus, post-
translational modifications of recombinant proteins have to be monitored during
production and documented for registration. Directly related to this topic, Chapter
9 shows how to monitor glycosylation in order to ensure product consistency.
Growing knowledge of the biological roles of protein modifications, on the one
hand, and the development and availability of sophisticated, sensitive analytical meth-
ods on the other hand, are already leading to increased interest in co- and posttransla-
tional modifications of proteins. Posttranslational Modifications of Proteins: Tools
for Functional Proteomics intends to serve as a practical guide for researchers working
in the field of protein structure–function relationships in general, in the rapidly growing
field of proteomics, as well as scientists in the pharmaceutical industries.
Christoph Kannicht
Contents
Preface .............................................................................................................v
Contributors.....................................................................................................ix
1 Assignment of Disulfide Bonds in Proteins by Chemical Cleavage
and Peptide Mapping by Mass Spectrometry
Jiang Wu and J. Throck Watson............................................................ 1
2 Carbohydrate Composition Analysis of Glycoproteins Using Highly
Sensitive Fluorescence Detection Methods
George N. Saddic, Mary Beth Ebert, Shirish T. Dhume,
and Kalyan R. Anumula..................................................................23
3 Enzymatical Hydrolysis of N-Glycans from Glycoproteins
and Fluorescent Labeling by 2-Aminobenzamide (2-AB)
Rolf Nuck..............................................................................................37
4 Separation of N-Glycans by HPLC
Martin Gohlke.......................................................................................45
5 Enzymatic Sequence Analysis of N-Glycans
Christoph Kannicht and Anke Flechner...........................................63
6 Immunological Detection of O-GlcNAc
Monika Rex-Mathes, Jürgen Koch, Sabine Werner, Lee S. Griffith,
and Brigitte Schmitz........................................................................73
7 Analysis of O-Glycosylation
Juan J. Calvete and Libia Sanz..........................................................89
8 Characterization of Site-Specific Glycosylation
Katalin F. Medzihradszky..................................................................101
9 Monitoring Glycosylation of Therapeutic Glycoproteins
for Consistency Using Highly Fluorescent Anthranilic Acid
Shirish T. Dhume, Mary Beth Ebert, George N. Saddic,
and Kalyan R. Anumula................................................................127
10 Metabolic Labeling and Structural Analysis
of Glycosylphosphatidylinositols from Parasitic Protozoa
Peter Gerold and Ralph T. Schwarz................................................143
11 Analysis of S-Acylation of Proteins
Michael Veit, Evgeni Ponimaskin, and Michael F. G. Schmidt......159
vii
viii Contents
12 Immunoblotting Methods for the Study of Protein Ubiquitination
Edward G. Mimnaugh and Leonard M. Neckers............................179
13 Analysis of Methylation and Acetylation in E. coli Ribosomal Proteins
Randy J. Arnold and James P. Reilly..............................................205
14 Identification of In Vivo Protein Phosphorylation Sites
with Mass Spectrometry
Jun Qin and Xiaolong Zhang............................................................211
15 Analysis of Tyrosine-O-Sulfation
Jens R. Bundgaard, Anders H. Johnsen, and Jens F. Rehfeld.....223
16 α-Amidated Peptides: Approaches for Analysis
Gregory P. Mueller and William J. Driscoll....................................241
17 γ-Glutamate and β-Hydroxyaspartate in Proteins
Francis J. Castellino, Victoria A. Ploplis, and Li Zhang...............259
18 Detection of isoAspartate Residues as a Posttranslational
Modification of Proteins and Peptides
Verne Schirch, Sonia Delle Fratte, and Martino di Salvo.............269
19 Lysine Hydroxylation and Crosslinking of Collagen
Mitsuo Yamauchi and Masashi Shiiba............................................277
20 Heterologous Expression in Endocrine Cells for Analysis
of Posttranslational Modifications
Jens R. Bundgaard............................................................................291
21 2D-Electrophoresis: Detection of Glycosylation and Influence
on Spot Pattern
Klemens Löster and Christoph Kannicht.......................................301
Index............................................................................................................317
Contributors
KALYAN R. ANUMULA • Analytical Sciences Department, SmithKline
Beecham, Research and Development, King of Prussia, PA
RANDY J. ARNOLD • Department of Chemistry, Indiana University,
Bloomington, IN
JENS R. BUNDGAARD •Department of Clinical Biochemistry, Rigshospitalet
Copenhagen University Hospital, Copenhagen, Denmark
JUAN J. CALVETE•Instituto de Biomedicina de Valencia, C. S. I. C.,
Valencia, Spain
FRANCIS J. CASTELLINO •Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, IN
SONIA DELLE FRATTE•Department of Biochemistry, Virginia Commonwealth
University, Richmond, VA
SHIRISH T. DHUME• Analytical Sciences Department, SmithKline Beecham,
Research and Development, King of Prussia, PA
MARTINO DI SALVO •Department of Biochemistry, Virginia Commonwealth,
University, Richmond, VA
WILLIAM J. DRISCOLL •Department of Anatomy, Physiology and Genetics,
Uniformed Services University of the Health Sciences, F. Edward Hébert
School of Medicine, Bethesda, MD
MARY BETH EBERT •Analytical Sciences Department, SmithKline Beecham,
Research and Development, King of Prussia, PA
ANKE FLECHNER •Institut für Molekularbiologie und Biochemie,
Freie Universität Berlin, Berlin, Germany
PETER GEROLD • Medizinisches Zentrum für Hygiene und Medizinische
Mikrobiologie, Philips-Universität Marburg, Marburg, Germany
MARTIN GOHLKE •Institut für Molekularbiologie und Biochemie,
Freie Universität Berlin, Berlin, Germany
LEE S. GRIFFITH•Department of Biochemistry, Institute of Animal Anatomy
and Physiology, University of Bonn, Bonn, Germany
ANDERS H. JOHNSEN •Department of Clinical Biochemistry, Rigshospitalet
Copenhagen University Hospital, Copenhagen, Denmark
ix