Characterization of Cell Glycocalyx with Mass Spectrometry Methods.

The cell membrane plays an important role in protecting the cell from its extracellular environment. As such, extensive work has been devoted to studying its structure and function. Crucial intercellular processes, such as signal transduction and immune protection, are mediated by cell surface glycosylation, which is comprised of large biomolecules, including glycoproteins and glycosphingolipids. Because perturbations in glycosylation could result in dysfunction of cells and are related to diseases, the analysis of surface glycosylation is critical for understanding pathogenic mechanisms and can further lead to biomarker discovery. Different mass spectrometry-based techniques have been developed for glycan analysis, ranging from highly specific, targeted approaches to more comprehensive profiling studies. In this review, we summarized the work conducted for extensive analysis of cell membrane glycosylation, particularly those employing liquid chromatography with mass spectrometry (LC-MS) in combination with various sample preparation techniques

Systems Glycobiology: Past, Present, and Future

Glycobiology is a glycan-based field of study that focuses on the structure, function, and biology of carbohydrates, and glycomics is a sub-study of the field of glycobiology that aims to define structure/function of glycans in living organisms. With the popularity of the glycobiology and glycomics, application of computational modeling expanded in the scientific area of glycobiology over the last decades. The recent availability of progressive Wet-Lab methods in the field of glycobiology and glycomics is promising for the impact of systems biology on the research area of the glycome, an emerging field that is termed “systems glycobiology.” This chapter will summarize the up-to-date leading edge in the use of bioinformatics tools in the field of glycobiology. The chapter provides basic knowledge both for glycobiologists interested in the application of bioinformatics tools and scientists of computational biology interested in studying the glycome

Integrated glycomics, proteomics, and glycoproteomics of human leukocytes and glioblastoma tissue microarrays

This thesis includes studies on N-, mucin type O-, and glycosaminoglycan (GAG)-linked glycosylation in human biospecimens. Glycosylation plays a central role in biological processes, including protein folding, immune surveillance, and regulation of cell growth. The structures of GAG are regulated in a tissue-specific manner. Heparan sulfate (HS) and chondroitin sulfate (CS) are the two types of GAGs targeted in this thesis. Human leukocytes express both CS and HS GAGs with CS being the more abundant type; however, little is known regarding the properties and structures of GAG chains, their ranges of variability among normal subjects, and changes in structure associated with disease conditions. We measured the relative and absolute disaccharides abundances of HS and CS for purified B, T, NK cells, monocytes, and polymorphonuclear leukocytes (PMNs) using size exclusion chromatography-mass spectrometry (SEC-MS). We found that all leukocytes express HS chains with levels of sulfation more similar to heparin than to organ-derived HS. In addition, CS abundances varied considerably in a leukocyte cell type specific manner. Therefore, our results established the ranges of GAG structures expressed on normal leukocytes as well as necessary for subsequent inquiry into disease conditions. Glioblastoma (GBM) accounts for 30% of human primary brain tumors. It is deadly and highly invasive. In past decades, most GBM research focused on pathophysiological changes in genome. There remains a dearth of knowledge regarding alterations in glycomics, glycoproteomics, and proteomics during GBM tumorigenesis. Therefore, we developed a comprehensive platform for high-throughput sample preparation with surface digestion for tissue microarrays, LC-MS/MS data dependent acquisition, and semi-automated data analysis to integrate glycomics, glycoproteomics, and proteomics for different grade of tumor and different subtypes of GBM. By analyzing GBM tissue microarrays, we found tumor grade and subtype specific changes to the expression of biomolecules. We also identified approximately 100 site-specific N- and mucin type O-glycosylations, the majority of which were previously unreported. Overall, our results improved the fundamental understandings about GBM pathogenesis.2018-11-02T00:00:00

High-resolution longitudinal N- and O-glycoprofiling of human monocyte-to-macrophage transition.

Protein glycosylation impacts the development and function of innate immune cells. The glycophenotypes and the glycan remodelling associated with the maturation of macrophages from monocytic precursor populations remain incompletely described. Herein, label-free porous graphitised carbon-liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) was employed to profile with high resolution the N- and O-glycome associated with human monocyte-to-macrophage transition. Primary blood-derived CD14+ monocytes were differentiated ex vivo in the absence of strong anti- and proinflammatory stimuli using a conventional 7-day granulocyte-macrophage colony-stimulating factor differentiation protocol with longitudinal sampling. Morphology and protein expression monitored by light microscopy and proteomics validated the maturation process. Glycomics demonstrated that monocytes and macrophages display similar N-glycome profiles, comprising predominantly paucimannosidic (Man1-3GlcNAc2Fuc0-1, 22.1-30.8%), oligomannosidic (Man5-9GlcNAc2, 29.8-35.7%) and α2,3/6-sialylated complex-type N-glycans with variable core fucosylation (27.6-39.1%). Glycopeptide analysis validated conjugation of these glycans to human proteins, while quantitative proteomics monitored the glycoenzyme expression levels during macrophage differentiation. Significant interperson glycome variations were observed suggesting a considerable physiology-dependent or heritable heterogeneity of CD14+ monocytes. Only few N-glycome changes correlated with the monocyte-to-macrophage transition across donors including decreased core fucosylation and reduced expression of mannose-terminating (paucimannosidic-/oligomannosidic-type) N-glycans in macrophages, while lectin flow cytometry indicated that more dramatic cell surface glycan remodelling occurs during maturation. The less heterogeneous core 1-rich O-glycome showed a minor decrease in core 2-type O-glycosylation but otherwise remained unchanged with macrophage maturation. This high-resolution glycome map underpinning normal monocyte-to-macrophage transition, the most detailed to date, aids our understanding of the molecular makeup pertaining to two vital innate immune cell types and forms an important reference for future glycoimmunological studies

Highlights of B/D‐HPP and HPP Resource Pillar Workshops at 12th Annual HUPO World Congress of Proteomics

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106985/1/pmic7698.pd

Characterization of Glycan and Glycoprotein by Mass Spectrometry for Therapeutic Drugs Development and Biomarker

Glycosylation is an essential post-translational modification of protein, which is involved in many biological processes including protein folding, immune response, protein-protein interaction and pathogens. Glycan exhibits complexity and dynamic changes in not only compositions of monosaccharides but also the linkages of them. Mass spectrometry is a powerful approach that provides a systematic and high-throughput analysis of protein glycosylation. In this dissertation, the mass spectrometry techniques have been utilized to analyze therapeutic glycoproteins and discover novel biomarkers of colon cancer. Numerous analytical chemistry techniques have been applied, including hydrophilic interaction enrichment of glycopeptide, solid phase based identification of O-glycosylation site, 18O labelling of N-glycosylation site, stepped collision energy induced glycopeptide dissociation, HCD/ETD alternative dissociation and CID based multi stage mass spectrometry. These advanced techniques have been applied in this dissertation to comprehensively understand the glycosylation of Coagulation factor V, discover the potential glycoprotein biomarkers of colon cancer and elucidate the unrevealed structure of pneumococcal polysaccharide vaccine

Glycoprotein analysis using protein microarrays and mass spectrometry

Protein glycosylation plays an important role in a multitude of biological processes such as cell–cell recognition, growth, differentiation, and cell death. It has been shown that specific glycosylation changes are key in disease progression and can have diagnostic value for a variety of disease types such as cancer and inflammation. The complexity of carbohydrate structures and their derivatives makes their study a real challenge. Improving the isolation, separation, and characterization of carbohydrates and their glycoproteins is a subject of increasing scientific interest. With the development of new stationary phases and molecules that have affinity properties for glycoproteins, the isolation and separation of these compounds have advanced significantly. In addition to detection with mass spectrometry, the microarray platform has become an essential tool to characterize glycan structure and to study glycosylation-related biological interactions, by using probes as a means to interrogate the spotted or captured glycosylated molecules on the arrays. Furthermore, the high-throughput and reproducible nature of microarray platforms have been highlighted by its extensive applications in the field of biomarker validation, where a large number of samples must be analyzed multiple times. This review covers a brief survey of the other experimental methodologies that are currently being developed and used to study glycosylation and emphasizes methodologies that involve the use of microarray platforms. This review describes recent advances in several options of microarray platforms used in glycoprotein analysis, including glycoprotein arrays, glycan arrays, lectin arrays, and antibody/lectin arrays. The translational use of these arrays in applications related to characterization of cells and biomarker discovery is also included. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 29:830–844, 2010Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77969/1/20269_ftp.pd

Developing an Integrative Glycobiology Workflow for the Identification of Disease Markers for Pancreatic Cancer

A deeper understanding of dysregulated glycosylation in pancreatic cancer can provide insights into disease mechanisms and the identification of novel disease markers. Recent improvements in mass spectrometry techniques have been instrumental in profiling biologically relevant tissue sections in order to identify disease marker candidates, but have either not yet been adopted for studying glycosylation or applied directly to pancreatic cancer. In the dissertation herein, new methods have been developed and adapted to the study of aberrant glycosylation in pancreatic cancer, with the ultimate goal of identifying novel disease marker candidates. For the first time, we describe a mass spectrometry imaging approach to study the localization of N-glycans. This technique demonstrated a histology-derived localization of N-glycans across tissue sections, with identifications displaying remarkable consistency with documented studies. Furthermore, the technique provides superior structural information compared to preexisting methodologies. In the analysis of diseased specimen, changes in glycosylation can be linked to aberrations in glycosyltransferase expression. When applied to pancreatic cancer in a high-throughput and high-dimensional analysis, panels of glycans displayed an improved ability to differentiate tumor from non-tumor tissues compared to current disease markers. Furthermore, the data suggest that glycosylation can identify premalignant lesions, as well as differentiate between malignant and benign conditions. These observations overcome significant limitations that hinder the efficacy of current disease markers. In an effort to link aberrant glycosylation to the modified protein, a subset of glycosylated proteins were enriched and analyzed by mass spectrometry to identify proteins that are integral to disease progression and can be probed for the early detection of pancreatic cancer. Known disease markers were among the glycoproteins identified, validating the utility of the enrichment and detection strategy outlined. This approach also differentiated the role of N- and O-glycosylation in antigen expression. Finally, we outline an integrated workflow that takes advantage of the unique capabilities of high resolution mass spectrometers. This workflow can capitalize on prior glycomic and proteomic experiments to provide a comprehensive analysis of dysregulated protein glycosylation in pancreatic cancer

Integrating glycomics, proteomics and glycoproteomics to understand the structural basis for influenza a virus evolution and glycan mediated immune interactions

Glycosylation modulates the range and specificity of interactions among glycoproteins and their binding partners. This is important in influenza A virus (IAV) biology because binding of host immune molecules depends on glycosylation of viral surface proteins such as hemagglutinin (HA). Circulating viruses mutate rapidly in response to pressure from the host immune system. As proteins mutate, the virus glycosylation patterns change. The consequence is that viruses evolve to evade host immune responses, which renders vaccines ineffective. Glycan biosynthesis is a non-template driven process, governed by stoichiometric and steric relationships between the enzymatic machinery for glycosylation and the protein being glycosylated. Consequently, protein glycosylation is heterogeneous, thereby making structural analysis and elucidation of precise biological functions extremely challenging. The lack of structural information has been a limiting factor in understanding the exact mechanisms of glycan-mediated interactions of the IAV with host immune-lectins. Genetic sequencing methods allow prediction of glycosylation sites along the protein backbone but are unable to provide exact phenotypic information regarding site occupancy. Crystallography methods are also unable to determine the glycan structures beyond the core residues due to the flexible nature of carbohydrates. This dissertation centers on the development of chromatography and mass spectrometry methods for characterization of site-specific glycosylation in complex glycoproteins and application of these methods to IAV glycomics and glycoproteomics. We combined the site-specific glycosylation information generated using mass spectrometry with information from biochemical assays and structural modeling studies to identify key glycosylation sites mediating interactions of HA with immune lectin surfactant protein-D (SP-D). We also identified the structural features that control glycan processing at these sites, particularly those involving glycan maturation from high-mannose to complex-type, which, in turn, regulate interactions with SP-D. The work presented in this dissertation contributes significantly to the improvement of analytical and bioinformatics methods in glycan and glycoprotein analysis using mass spectrometry and greatly advances the understanding of the structural features regulating glycan microheterogeneity on HA and its interactions with host immune lectins