posttranslational modifications of proteins

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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 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 v 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 vi Preface (in Chapters 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 R ANDY J. ARNOLD • Department of Chemistry, Indiana University, Bloomington, IN J ENS R. BUNDGAARD • Department of Clinical Biochemistry, Rigshospitalet Copenhagen University Hospital, Copenhagen, Denmark J UAN J. CALVETE • Instituto de Biomedicina de Valencia, C. S. I. C., Valencia, Spain F RANCIS J. CASTELLINO • Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN S ONIA DELLE FRATTE • Department of Biochemistry, Virginia Commonwealth University, Richmond, VA S HIRISH T. DHUME • Analytical Sciences Department, SmithKline Beecham, Research and Development, King of Prussia, PA M ARTINO DI SALVO • Department of Biochemistry, Virginia Commonwealth, University, Richmond, VA W ILLIAM J. DRISCOLL • Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, F. Edward Hébert School of Medicine, Bethesda, MD M ARY BETH EBERT • Analytical Sciences Department, SmithKline Beecham, Research and Development, King of Prussia, PA A NKE FLECHNER • Institut für Molekularbiologie und Biochemie, Freie Universität Berlin, Berlin, Germany P ETER GEROLD • Medizinisches Zentrum für Hygiene und Medizinische Mikrobiologie, Philips-Universität Marburg, Marburg, Germany M ARTIN GOHLKE • Institut für Molekularbiologie und Biochemie, Freie Universität Berlin, Berlin, Germany L EE S. GRIFFITH • Department of Biochemistry, Institute of Animal Anatomy and Physiology, University of Bonn, Bonn, Germany A NDERS H. JOHNSEN • Department of Clinical Biochemistry, Rigshospitalet Copenhagen University Hospital, Copenhagen, Denmark ix [...]... YAMAUCHI • Dental Research Center, University of North Carolina, Chapel Hill, NC LI ZHANG • Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN XIAOLONG ZHANG • Department of Biochemistry and Department of Cell Biology, Baylor College of Medicine, Houston, TX Assignment of Disulfide Bonds 1 1 Assignment of Disulfide Bonds in Proteins by Chemical Cleavage and Peptide Mapping... reduction of the disulfide bond The analysis of a protein containing multiple disulfide bonds requires some means of reducing a particular disulfide bond and applying the cyanylation/ cleavage methodology to corresponding pair of nascent cysteines (11) The technique of partial reduction developed by Gray (14,15) provides a convenient means of preparing a mixture of partially reduced isoforms of the parent... chromatography of proteins Anal Biochem 103, 1–25 20 Rubinstein, M (1979) Preparative high-performance liquid partition chromatography of proteins Anal Biochem 98, 1–7 Analysis of Glycoproteins 23 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 1 Introduction Proteins with... of oligosaccharides, which is described in Chapter 9 of this book, and determination of carbohydrate composition Carbohydrate composition analysis of glycoproteins is similar to amino acid analysis of proteins Just as an accurate amino acid composition is critical to protein-structure determination and identification by database searching, the accurate determination of the carbohydrate composition of. .. Assignment of Disulfide Bonds 1 26 40 58 65 13 72 84 95 110 124 Scheme 1 Disulfide bond linkage of Ribonuclease A Figure 6A–D are MALDI spectra of peptide mixtures resulting from cleavage of one of the four singly reduced/cyanylated isoforms of ribonuclease A corresponding to HPLC peaks 1–4 in Fig 5, respectively Table 1 lists the calculated and observed m/z values for fragments resulting from the cleavage of. .. rate of disulfide reduction may vary, the extent of reduction should be monitored for individual proteins From the limited number of proteins we have examined, it appears that an equivalent of TCEP for the cysteine content is a good initial stoichiometry The extent of reduction may be readily adjusted by controlling the reaction time and temperature 20 Wu and Watson 7 A fivefold molar excess of CDAP... posttranslational modifications in proteins Disulfide bonds play an important role in folding/refolding processes and in maintaining the three-dimensional structure of proteins The determination of disulfide-bond linkage is therefore an integral part of structural characterization of proteins (2) Conventional methodology for determining disulfide linkage involves the cleavage of the protein backbone between... Department of Biochemistry and Department of Cell Biology, Baylor College of Medicine, Houston, TX JENS F REHFELD • Department of Clinical Biochemistry, Rigshospitalet Copenhagen University Hospital, Copenhagen, Denmark JAMES P REILLY • Department of Chemistry, Indiana University, Bloomington, IN MONIKA REX-MATHES • Department of Biochemistry, Institute of Animal Anatomy and Physiology, University of Bonn,... the confirmation of the assignment in this case The HPLC peaks 1 and 2 in Fig 5 were not resolved completely This is reflected in the MALDI spectra (Fig 6A,B) of the cleavage products of these two fractions, each of which carries small fragments corresponding to the cleavage products of the other fraction The unambiguous assignment of the respective disulfide bond pairs in the presence of another isomer... KOCH • Department of Biochemistry, Institute of Animal Anatomy and Physiology, University of Bonn, Bonn, Germany KLEMENS LÖSTER • Arevia GmbH, Berlin, Germany KATALIN F MEDZIHRADSZKY • Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA EDWARD G MIMNAUGH • National Cancer Institute, NIH, Rockville, MD GREGORY P MUELLER • Department of Anatomy, Physiology . Kannicht VOLUME 194 Posttranslational Modifications of Proteins Tools for Functional Proteomics Tools for Functional Proteomics Posttranslational Modifications of Proteins Edited by Christoph Kannicht Posttranslational. 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. 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