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(Uncorrected OCR) Abstract of thesis entitled MASS SPECTROMETRIC ANALYSIS OF SELECTED GLYCOPROTEINS Submitted by Chan Chun Yu for the degree of Master of Philosophy at The University of Hong Kong in February 2005 Fetuin from fetal calf serum (Sigma, F6131), a well-characterized glycoprotein that has a molecular weight of 48.4 kDa and 3 N-glycosylation sites, was used as a prototypical protein to develop mass spectrometric approaches toward the identification and sequence elucidation of the N-glycosylation sites of proteins. Fetuin was digested using trypsin (porcine) and deglycosylated using peptide N-glycosidase F (PNGase-F) prior to matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. For peptide mass fingerprinting, the experimental peak list obtained from the MALDI-TOF mass spectrum of the sample was compared with the theoretical peak list obtained from the National Center for Biotechnology Information sequence database. The identification of each N-glycosylation site was based on the 0.98-Da mass increase observed upon hydrolysis by the amidase PNGase-F of the glycosylamine linkage of each N-glycosylated asparagine, which formed an aspartic acid residue. The identities of the glycopeptides were verified through isotopic pattern analysis using the proposed amino acid composition and the ISOPRO shareware program. All the 3 potential N-glycosylation sites of fetuin were identified successfully in accordance with published results. The peptide sequence coverage was 75%. The developed strategy was then applied to map the N-glycosylation sites of the severe acute respiratory syndrome spike S glycoprotein, a novel glycoprotein that requires a more complex analysis for its total 23 potential N-glycosylation sites. The proposed peptide sequence and its potential N-glycosylation sites were elucidated using tandem mass spectrometry under low-energy collision-induced dissociation. The partial peptide sequence was determined by.
This volume presents methods used for the analysis of glycoproteins at different levels—intact, subunit, glycopeptide, and monosaccharide--, and discusses and solves most analytical challenges that a scientist working on glycoproteins may come across. The chapters in this book cover topics such as the role of glycosylation on the properties of therapeutic glycoproteins; different analytical methods to characterize glycosylation, from the intact proteins to the glycan level, for both N-linked and O-linked glycoproteins; mass spectrometry imaging methodology for glycosylation analysis in tissues; approaches to characterizing glycosylation on cultured cells; and the use of cloud computing to deploy mass spectrometry data analysis. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Mass Spectrometry of Glycoproteins: Methods and Protocols is a valuable resource for scientists interested in learning more about this developing field.
As one of the most extensive and important protein post-translational modifications, glycosylation plays a vital role in regulating organisms and is associated with various physiological and pathological processes. Recently, researchers have focused on the need to characterize protein glycosylation sites, structures, and their degree of modification, to better understand their biological functions while also looking for potential biomarkers for diagnosis and treatment of disease. Mass spectrometry (MS) is one of the most powerful tools used to study biomolecules including glycoproteins and glycans. With the continuous development of glycoproteomics and glycomics based on MS analysis, more techniques have evolved and contribute to understanding the structure and function of glycoproteins and glycans. This book reviews advancements achieved in MS-based glycoproteomic analysis, including a wide range of analytical methodologies and strategies involved in selective enrichment; as well as qualitative, quantitative, and data analysis, together with their clinical applications. Significant examples are discussed to illustrate the principles, laboratory protocols, and advice for key implementation to ensure successful results. Mass Spectrometry–Based Glycoproteomics and Its Clinic Application will serve as a valuable resource to elucidate new techniques and their applications for students, postdocs, and researchers working in proteomics, glycoscience, analytical chemistry, biochemistry, and clinical medicine. Editor: Haojie Lu is a professor at Fudan University, specializing in proteomics based on mass spectrometry with particular emphasis on novel technologies for separation and identification of low-abundant proteins and post-translationally modified proteins (including glycosylation), as well as relative and absolute quantification methods for proteomics.
PROVIDES STRATEGIES AND CONCEPTS FOR UNDERSTANDING CHEMICAL PROTEOMICS, AND ANALYZING PROTEIN FUNCTIONS, MODIFICATIONS, AND INTERACTIONS—EMPHASIZING MASS SPECTROMETRY THROUGHOUT Covering mass spectrometry for chemical proteomics, this book helps readers understand analytical strategies behind protein functions, their modifications and interactions, and applications in drug discovery. It provides a basic overview and presents concepts in chemical proteomics through three angles: Strategies, Technical Advances, and Applications. Chapters cover those many technical advances and applications in drug discovery, from target identification to validation and potential treatments. The first section of Mass Spectrometry-Based Chemical Proteomics starts by reviewing basic methods and recent advances in mass spectrometry for proteomics, including shotgun proteomics, quantitative proteomics, and data analyses. The next section covers a variety of techniques and strategies coupling chemical probes to MS-based proteomics to provide functional insights into the proteome. In the last section, it focuses on using chemical strategies to study protein post-translational modifications and high-order structures. Summarizes chemical proteomics, up-to-date concepts, analysis, and target validation Covers fundamentals and strategies, including the profiling of enzyme activities and protein-drug interactions Explains technical advances in the field and describes on shotgun proteomics, quantitative proteomics, and corresponding methods of software and database usage for proteomics Includes a wide variety of applications in drug discovery, from kinase inhibitors and intracellular drug targets to the chemoproteomics analysis of natural products Addresses an important tool in small molecule drug discovery, appealing to both academia and the pharmaceutical industry Mass Spectrometry-Based Chemical Proteomics is an excellent source of information for readers in both academia and industry in a variety of fields, including pharmaceutical sciences, drug discovery, molecular biology, bioinformatics, and analytical sciences.
Glycoproteins are central to numerous cellular processes and are among the most structurally complex biomolecules in nature. This unique complexity stems from variability in complex oligosaccharides that are located throughout the protein, a feature that is profoundly important for regulating biomolecular interactions but also makes glycoproteins difficult to study. As such, glycoprotein analysis entails a range of techniques to bridge the knowledge gap between glycoprotein structure and biological function. This book serves as an authoritative guide to glycoprotein analysis, written by internationally recognised experts in the field and discussed in the context of real-world applications across the life sciences. It provides a wide-ranging assessment of the modern methods, from those used to characterise glycoprotein structure, to approaches proficient in uncovering the molecular mechanisms by which they function as well as those capable of measuring structural dynamics and macromolecular assembly. These methods differ to a large extent and include mass spectrometry, glycan/lectin arrays, nuclear magnetic resonance, infrared spectroscopy, scanning probe microscopy and high-performance liquid chromatography. Equally important are computational techniques, including molecular dynamics and bioinformatics, which are also covered and discussed in the wider context of glycoprotein analysis. Glycobiology is indeed a rapidly growing field and the development of advanced tools for glycoproteins analysis has been enabled by researchers from different backgrounds working to overcome long-standing analytical challenges and biological questions involving glycosylation. This book is intended to aid academic and professional researchers at various levels of their career to gain a deeper appreciation of cutting-edge methods in glycoprotein analysis and their applications in biomolecular research, biotherapeutic development, structural biology and biophysical chemistry.
This comprehensive new resource in Springer s Methods in Molecular Biology series features contributions from leading researchers who provide expert advice and reproducible, cutting-edge protocols for examining glycoproteins through mass spectrometry."
Covers all major modifications, including phosphorylation, glycosylation, acetylation, ubiquitination, sulfonation and and glycation Discussion of the chemistry behind each modification, along with key methods and references Contributions from some of the leading researchers in the field A valuable reference source for all laboratories undertaking proteomics, mass spectrometry and post-translational modification research
Mass Spectrometry: Techniques for the Structural Characterization of Glycans presents new methods for conducting detailed carbohydrate qualitative analysis—arming analytical chemists, pharmaceutical scientists, and food scientists with a quick reference that will allow them to determine the structures of carbohydrates molecules. As there is a need in the scientific community for content specific to structural determination and analysis of new glycoprotein drug, and because structure-activity analysis requires a structural determination of the N- and O-linked oligosaccharides linked to glycol-proteins, this book provides the relevant research that are necessary for advances and new outcomes in this area of study. - Authored by an analytical chemist with more than 30 years of experience in research and industry - Serves as a quick reference in mass spectral analysis and carbohydrates - Includes more than 60 figures to aid in the retention of key concepts