Chemical Glycoproteomics For Identification And Discovery Of Glycoprotein Alterations In Human Cancer

Changes in glycosylation have long been appreciated to be part of the cancer phenotype; sialylated glycans are found at elevated levels on many types of cancer and have been implicated in disease progression. However, the specific glycoproteins that contribute to cell surface sialylation are not well characterized, specifically in bona fide human cancer. Metabolic and bioorthogonal labeling methods have previously enabled enrichment and identification of sialoglycoproteins from cultured cells and model organisms. The goal of this work was to develop technologies that can be used for detecting changes in glycoproteins in clinical models of human cancer. In Chapter 1 of this dissertation, I present an overview of the structures and functions of glycans and their relationship to cancer progression. I also discuss applications of in vivo bioorthogonal labeling in model organisms and how in humans, the significant regulatory and ethical barriers associated with introducing chemically altered sugars into people have hindered it. Finally, I review mass spectrometry-based proteomics and how it can be applied to clinical glycoproteomics. In Chapter 2, I demonstrate the first application of this bioorthogonal labeling in a glycoproteomics platform applied to human tissues cultured ex vivo. Both normal and cancerous prostate tissues were sliced and cultured in the presence of functionalized derivatives of N-acetyl mannosamine, the sialic acid biosynthetic precursor. Chemical biotinylation followed by enrichment and mass spectrometry led to the identification of glycoproteins that were found at elevated levels or uniquely in cancerous prostate tissue. This work therefore extends the use of bioorthogonal labeling strategies to problems of human clinical relevance. Secretome proteins play important roles in regulation of many physiological processes and show utility as potential biomarkers and for noninvasive diagnostics and treatment monitoring. In Chapter 3, I discuss a platform for identifying sialoglycoproteins that were secreted in the conditioned media from bioorthogonally labeled human prostate tissue slice cultures. This platform could be used to identify disease biomarkers in a faithful clinical model of human disease. Mutations in granulocyte colony-stimulating factor 3 receptor (CSF3R), also known as G-CSFR, occur in the majority of patients with chronic neutrophilic leukemia (CNL) and are more rarely present in other kinds of leukemia. In Chapter 4, I discuss novel variants in CSF3R at asparagine residue N610, one of which was germline. Interestingly, these N610 substitutions are potently oncogenic and result in ligand-independent receptor activation. They confer activation of the JAK-STAT signaling pathway and concurrent sensitivity to JAK kinase inhibitors. The N610 residue is part of a consensus N-linked glycosylation motif in the receptor. Detailed mass spectrometry analysis demonstrates that this site is occupied by both complex and complex bisecting glycans. Further analysis demonstrates that N610 is the primary site of sialylation of the receptor. This study demonstrates that membrane-proximal N-linked glycosylation is critical for maintaining the ligand dependence of the receptor. Furthermore, it expands the repertoire of potently oncogenic mutations in CSF3R that are therapeutically targetable.

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.

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.

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.

This volume provides a comprehensive understanding of the enigmatic identity of the glycome, a complex but important area of research that has been largely ignored due to its complexity. The authors thoroughly deal with almost all aspects of the glycome, i.e., elucidation of the glycan identity enigma and its role in regulation of the cellular process, and in disease etiology. The book bridges the knowledge gap in understanding the glycome, from being a cell signature to its applications in disease etiology. In addition, it details many of the major insights regarding the possible role of the glycome in various diseases as a therapeutic marker. The book systematically covers the major aspects of the glycome, including the significance of substituting the diverse monosaccharide units to glycoproteins, the role of glycans in disease pathologies, and the challenges and advances in glycobiology. The authors stress the significance and huge encoding power of carbohydrates as well as provide helpful insights in framing the bigger picture. The Glycome: Understanding the Diversity and Complexity of Glycobiology details state-of-the-art developments and emerging challenges of glycome biology, which are going to be key areas of future research, not only in the glycobiology field but also in pharmaceutics.

Advances in Cancer Research provides invaluable information on the exciting and fast-moving field of cancer research. Here, once again, outstanding and original reviews are presented on a variety of topics. Provides information on cancer research Outstanding and original reviews Suitable for researchers and students

This book provides a comprehensive overview of the current limitations and unmet needs in Hepatocellular Carcinoma (HCC) diagnosis, treatment, and prevention. It also provides newly emerging concepts, approaches, and technologies to address challenges. Topics covered include changing landscape of HCC etiologies in association with health disparities, framework of clinical management algorithm, new and experimental modalities of HCC diagnosis and prognostication, multidisciplinary treatment options including rapidly evolving molecular targeted therapies and immune therapies, multi-omics molecular characterization, and clinically relevant experimental models. The book is intended to assist collaboration between the diverse disciplines and facilitate forward and reverse translation between basic and clinical research by providing a comprehensive overview of relevant areas, covering epidemiological trend and population-level patient management strategies, new diagnostic and prognostic tools, recent advances in the standard care and novel therapeutic approaches, and new concepts in pathogenesis and experimental approaches and tools, by experts and opinion leaders in their respective fields. By thoroughly and concisely covering whole aspects of HCC care, Hepatocellular Carcinoma serves as a valuable reference for multidisciplinary readers, and promotes the development of personalized precision care strategies that lead to substantial improvement of disease burden and patient prognosis in HCC.

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

Provides timely, comprehensive coverage of in vivo chemical reactions within live animals This handbook summarizes the interdisciplinary expertise of both chemists and biologists performing in vivo chemical reactions within live animals. By comparing and contrasting currently available chemical and biological techniques, it serves not just as a collection of the pioneering work done in animal-based studies, but also as a technical guide to help readers decide which tools are suitable and best for their experimental needs. The Handbook of In Vivo Chemistry in Mice: From Lab to Living System introduces readers to general information about live animal experiments and detection methods commonly used for these animal models. It focuses on chemistry-based techniques to develop selective in vivo targeting methodologies, as well as strategies for in vivo chemistry and drug release. Topics include: currently available mouse models; biocompatible fluorophores; radionuclides for radiodiagnosis/radiotherapy; live animal imaging techniques such as positron emission tomography (PET) imaging; magnetic resonance imaging (MRI); ultrasound imaging; hybrid imaging; biocompatible chemical reactions; ligand-directed nucleophilic substitution chemistry; biorthogonal prodrug release strategies; and various selective targeting strategies for live animals. -Completely covers current techniques of in vivo chemistry performed in live animals -Describes general information about commonly used live animal experiments and detection methods -Focuses on chemistry-based techniques to develop selective in vivo targeting methodologies, as well as strategies for in vivo chemistry and drug release -Places emphasis on material properties required for the development of appropriate compounds to be used for imaging and therapeutic purposes in preclinical applications Handbook of In Vivo Chemistry in Mice: From Lab to Living System will be of great interest to pharmaceutical chemists, life scientists, and organic chemists. It will also appeal to those working in the pharmaceutical and biotechnology industries.