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Mass spectrometry (MS) has evolved as a powerful tool for glycosylation analysis benefiting from its high speed, high resolution and sensitivity. It enables detailed structure characterization of glycans, glycosylation site and the peptide sequences of complex biological samples in a high-throughput. This dissertation is dedicated to developing MS-based analytical methods for the structure characterization and quantitative analysis of both released glycans and intact glycopeptides utilizing a bottom-up approach. We started by developing a hydrophilic interaction liquid chromatography (HILIC) coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) platform for improved quantitative analysis of N-glycans compared to MALDI-MS. Faced with the challenges of N-glycan structure complexity, capillary electrophoresis (CE) emerges as a robust separation technique affording very small amount of sample consumption, fast separation speed, and high resolving power. Therefore, we developed a CE-ESI-MS/MS based 6-plex aminoxy tandem mass tag (TMT) labeling method for quantitative analysis of protein N-glycosylation analysis from human serum, providing the ability of N-glycan quantitation across up to six samples. Improved resolution and quantification accuracy of the labeled human milk oligosaccharides (HMOs) isomers was also achieved by coupling CE with traveling wave ion mobility (TWIM)-CID-MS/MS. The advantage of the glycan-focused approach (glycomics) is that the wealthy structure information and glycan isomer separation could be achieved; however, this approach resulted in loss of the glycosylation site-specific information. Therefore, we developed a powerful glycoproteomics workflow for system-wide structure characterization and quantitation of intact glycopeptides. Improvements of this workflow include improved glycoprotein extraction, sequential enrichment strategies for improved N-/O- glycopeptide enrichment, hybrid novel fragmentation technique electron transfer and higher-energy collision dissociation (EThcD), multiplex quantitation enabled by our custom-made isobaric N, N-dimethyl leucine (DiLeu) tags, and automated FDR-based large-scale data analysis by Byonic. We have successfully applied this workflow to analyze complex biological samples for disease-related glyco-alteration study, including the glycosylation alteration in PKM2 knockout breast cancer cells vs. parental cells and glycosylation pattern alteration in AD patients.
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.
The development of analytical technologies to investigate the glycoproteome of clinical relevant samples has improved over the last 10 years. These new developments aim to improve the identification and quantification of disease-specific glyco-biomarkers, which are present at low amounts in biological matrices. Glyco-biomakers have the potential to significantly contribute to cancer discovery studies in specific areas such as; early diagnosis, prognosis, monitor cancer recurrence and improve the low survival rate of cancer. In this thesis, we focused on the development and application of novel liquid affinity chromatography fractionation platforms integrated with nano-LC-MS/MS to characterize and quantify the glycoproteome as well as selected glyco-biomarker candidates of cancer samples. In chapter 1, brief background information covering glycoproteomics and glyco-biomarker discovery studies is presented. Specifically, protein glycosylation process and how the field of 'omics', which includes glycoproteomics, have revolutionized clinical glyco-biomarkers discovery are discussed. Further, various disease models, current sample fractionation strategies and analytical methodologies involved in glyco-biomarker development pipeline and their significance as well as their short falls are described. Reviewing biomarker validation and current bio-infomatics tools utilized in glycoproteomics discovery studies concludes chapter 1. Chapter 2 details the development of a novel multi-dimensional affinity liquid chromatography fractionation approach that combines the depletion of the top 12 abundant proteins and multi-lectin fractionation of the human plasma. Evaluating and validating the reproducibility, specificity and overall recoveries of the platform demonstrated the suitability of the developed method in glyco-biomarker discovery studies of clinical samples. After establishing this robust platform, it was applied in chapter 3 to comprehensively study the global glycoproteome profile of clear cell renal cell carcinoma plasma (ccRCC) samples to identify and characterize potential biomarkers for early detection of the disease. During this study, protein abundance alterations as well as glycan shifts were investigated to understand the sub-proteome of ccRCC. Chapter 4 focuses on the structural characterization of a glycoprotein (clusterin) that was identified during the ccRCC biomarker discovery studies. Clusterin has been implicated in ccRCC cancer progression however; its structure and biological function(s) are not yet well defined. Therefore, to have more structural insights into clusterin, the protein was immuno-affinity purified from ccRCC plasma followed by tandem mass spectrometry to profile glycoforms, "N"-glycosylation sites and quantify glycan amounts. We discovered that the levels of bi-antennary digalactosylated disialylated (A2G2S2) and core fucosylated bi-antennary digalactosylated disialylated (FA2G2S2) glycans differed significantly in the plasma of patients before and after curative nephrectomy of localized ccRCC. In chapter 5, a multi-lectin affinity chromatography platform previously developed in our laboratory was optimized and applied to investigate glycoproteins and non-glycoproteins present in pancreatic cyst fluid samples. This study was aimed at identifying potential candidate markers for early detection of malignant cyst (pancreatic cancer precursor). Our data showed the identification of proteins with significant differential expression in mucinous cysts (malignant cyst) compared to non-mucinous cysts (benign) of which one protein (periostin) associated with cancer progression was confirmed by immunoblotting assay. In the final chapter (chapter 6), we summarize and conclude our findings in this work and provide our perspective on the potential of glycoproteins in glyco-biomarker discovery studies.
This book summarizes recent advances in antibody glycosylation research. Covering major topics relevant for immunoglobulin glycosylation - analytical methods, biosynthesis and regulation, modulation of effector functions - it provides new perspectives for research and development in the field of therapeutic antibodies, biomarkers, vaccinations, and immunotherapy. Glycans attached to both variable and constant regions of antibodies are known to affect the antibody conformation, stability, and effector functions. Although it focuses on immunoglobulin G (IgG), the most explored antibody in this context, and unravels the natural phenomena resulting from the mixture of IgG glycovariants present in the human body, the book also discusses other classes of human immunoglobulins, as well as immunoglobulins produced in other species and production systems. Further, it reviews the glycoanalytical methods applied to antibodies and addresses a range of less commonly explored topics, such as automatization and bioinformatics aspects of high-throughput antibody glycosylation analysis. Lastly, the book highlights application areas ranging from the ones already benefitting from antibody glycoengineering (such as monoclonal antibody production), to those still in the research stages (such as exploration of antibody glycosylation as a clinical or biological age biomarker), and the potential use of antibody glycosylation in the optimization of vaccine production and immunization protocols. Summarizing the current knowledge on the broad topic of antibody glycosylation and its therapeutic and biomarker potential, this book will appeal to a wide biomedical readership in academia and industry alike. Chapter 4 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.
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."
This book, now in an extensively revised second edition, provides a comprehensive summary of the latest knowledge regarding glycosignals and a thorough analysis of their involvement in not only cancers but also other refractory conditions such as chronic inflammatory disorders. Many relevant topics are covered, including the search for novel tumor epitopes related to carbohydrates, the assembly of glycoconjugates, the modulation of signaling pathways by glycosylation, and interactions between complex carbohydrates and their recognition molecules. The role of various research approaches, for example advanced mass spectrometry, high-resolution imaging, and bioinformatics, is closely examined, and the results of novel therapeutic trials targeting glycosignals are discussed. The book will be essential reading for students and young researchers with an interest in glycoscience. In presenting new results and approaches and identifying areas for future research, it will also be of benefit for specialists in the field.
A new focus on glycoscience, a field that explores the structures and functions of sugars, promises great advances in areas as diverse as medicine, energy generation, and materials science, this report finds. Glycans-also known as carbohydrates, saccharides, or simply as sugars-play central roles in many biological processes and have properties useful in an array of applications. However, glycans have received little attention from the research community due to a lack of tools to probe their often complex structures and properties. Transforming Glycoscience: A Roadmap for the Future presents a roadmap for transforming glycoscience from a field dominated by specialists to a widely studied and integrated discipline, which could lead to a more complete understanding of glycans and help solve key challenges in diverse fields.
Sugar chains (glycans) are often attached to proteins and lipids and have multiple roles in the organization and function of all organisms. "Essentials of Glycobiology" describes their biogenesis and function and offers a useful gateway to the understanding of glycans.
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