The glycoproteome is an intricate and diverse component of a cell, and it plays a key role in the definition of the interface between that cell and the rest of its world. Methods for studying the glycoproteome have been developed for released glycan glycomics and site-localized bottom-up glycoproteomics using liquid chromatography-coupled mass spectrometry and tandem mass spectrometry (LC-MS/MS), which is itself a complex problem. Algorithms for interpreting these data are necessary to be able to extract biologically meaningful information in a high throughput, automated context. Several existing solutions have been proposed but may be found lacking for larger glycopeptides, for complex samples, different experimental conditions, different instrument vendors, or even because they simply ignore fundamentals of glycobiology. I present a series of open algorithms that approach the problem from an instrument vendor neutral, cross-platform fashion to address these challenges, and integrate key concepts from the underlying biochemical context into the interpretation process. In this work, I created a suite of deisotoping and charge state deconvolution algorithms for processing raw mass spectra at an LC scale from a variety of instrument types. These tools performed better than previously published algorithms by enforcing the underlying chemical model more strictly, while maintaining a higher degree of signal fidelity. From this summarized, vendor-normalized data, I composed a set of algorithms for interpreting glycan profiling experiments that can be used to quantify glycan expression. From this I constructed a graphical method to model the active biosynthetic pathways of the sample glycome and dig deeper into those signals than would be possible from the raw data alone. Lastly, I created a glycopeptide database search engine from these components which is capable of identifying the widest array of glycosylation types available, and demonstrate a learning algorithm which can be used to tune the model to better understand the process of glycopeptide fragmentation under specific experimental conditions to outperform a simpler model by between 10% and 15%. This approach can be further augmented with sample-wide or site-specific glycome models to increase depth-of-coverage for glycoforms consistent with prior beliefs.

Glycoproteins, and their subset proteoglycans, are an important group of molecules in joint tissues, providing crucial functions such as cartilage structural integrity and lubrication at cartilage surfaces. The functionality of these glycoproteins is attributable to their oligosaccharide components, however surprisingly little is known about their fine structural details. With the use of glycoproteomic methods, this thesis presents the development and incorporation of mass spectrometric, biochemical and immunological methods to elucidate glycoprotein structures in synovial fluids, chondrocytes and synoviocytes in order to provide insight into how their structures may contribute to their functions. Initially, anion exchange chromatography was used to extract the acidic fraction containing glycoproteins and proteoglycans in arthritic synovial fluid (SF) samples, followed by proteomic analysis to identify the main glycoproteins in 1D-SDS-PAGE gels. To complement these findings, an in-gel enzymatic digest method for glycosaminoglycan (GAG) and oligosaccharide analysis was developed for analysis of glycoproteins by graphitised carbon liquid chromatography mass spectrometry (LC-MS). Further characterization of the major glycoprotein, lubricin, was pursued by investigating its interactions with the surrounding extracellular matrix (ECM) from its cellular sources and characterising the secreted lubricin with Western blot and proteomic analysis. Finally, the graphitised carbon LC-MS method was applied to analyse the overall glycosylation profiles of lubricin. The major glycoprotein found in arthritic synovial fluid was lubricin, as identified by peptide LC-MS and Western blot. Graphitised carbon LC-MS identified the major chondroitin sulfate (CS) repeat region disaccharides and linkage region oligosaccharides of aggrecan with confirmation through tandem mass spectra and Western blots using CS linkage region stub antibodies. Application of this method to lubricin led to the discovery of O-linked oligosaccharide structures which were previously undescribed for lubricin. A higher proportion of sialylated oligosaccharide structures were detected in the rheumatoid arthritis (RA) samples compared to the osteoarthritic (OA) samples, which signifies a diagnostic difference between these diseases. Sulfated oligosaccharide structures were also detected on synovial fluid lubricin, correlated with Western blot reactivity with the MECA-79 antibody, thus suggesting a role for lubricin in inflammation. Overall the results demonstrated that glycosylation structure indicates additional functional properties for the glycoproteins such as lubricin.

Glycoproteins, and their subset proteoglycans, are an important group of molecules in joint tissues, providing crucial functions such as cartilage structural integrity and lubrication at cartilage surfaces. The functionality of these glycoproteins is attributable to their oligosaccharide components, however surprisingly little is known about their fine structural details. With the use of glycoproteomic methods, this thesis presents the development and incorporation of mass spectrometric, biochemical and immunological methods to elucidate glycoprotein structures in synovial fluids, chondrocytes and synoviocytes in order to provide insight into how their structures may contribute to their functions. Initially, anion exchange chromatography was used to extract the acidic fraction containing glycoproteins and proteoglycans in arthritic synovial fluid (SF) samples, followed by proteomic analysis to identify the main glycoproteins in 1D-SDS-PAGE gels. To complement these findings, an in-gel enzymatic digest method for glycosaminoglycan (GAG) and oligosaccharide analysis was developed for analysis of glycoproteins by graphitised carbon liquid chromatography mass spectrometry (LC-MS). Further characterization of the major glycoprotein, lubricin, was pursued by investigating its interactions with the surrounding extracellular matrix (ECM) from its cellular sources and characterising the secreted lubricin with Western blot and proteomic analysis. Finally, the graphitised carbon LC-MS method was applied to analyse the overall glycosylation profiles of lubricin. The major glycoprotein found in arthritic synovial fluid was lubricin, as identified by peptide LC-MS and Western blot. Graphitised carbon LC-MS identified the major chondroitin sulfate (CS) repeat region disaccharides and linkage region oligosaccharides of aggrecan with confirmation through tandem mass spectra and Western blots using CS linkage region stub antibodies. Application of this method to lubricin led to the discovery of O-linked oligosaccharide structures which were previously undescribed for lubricin. A higher proportion of sialylated oligosaccharide structures were detected in the rheumatoid arthritis (RA) samples compared to the osteoarthritic (OA) samples, which signifies a diagnostic difference between these diseases. Sulfated oligosaccharide structures were also detected on synovial fluid lubricin, correlated with Western blot reactivity with the MECA-79 antibody, thus suggesting a role for lubricin in inflammation. Overall the results demonstrated that glycosylation structure indicates additional functional properties for the glycoproteins such as lubricin.

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.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第25008号 / 医科博第155号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 伊藤 貴浩, 教授 藤田 恭之, 教授 伊藤 能永 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

N-linked glycosylation is the most common post-translational modification (PTM) of proteins that exist in nature. N-glycosylation and change in cells serve as a criterion to monitor the activity of developmental stages and diseases severity. Currently, there is an increasing application of mass spectrometry on glycoprotein for malicious, chronic or acute diseases, such as cancers, rheumatoid arthritis (RA) or influenza. In this dissertation, several mass spectrometric assays have been utilized to, quantitatively and qualitatively, characterize protein N-glycosylation at the glycan, glycopeptide and peptide levels. The goals are to identify serum-based RA biomarker (Chapter 2), or to determine possible glycan structures from monoclonal antibody (Chapter 3), or comprehensively to study one influenza glycoprotein, hemagglutinin (Chapter 4). In Chapter 2, LC-MS/MS with CID as MS 2 is the primary technique that is applied to collect raw data for RA biomarker screening; western blot is the verification method for newfound biomarkers. This mass spectrometry based comparative analysis of N-glycoprotein in RA and healthy patients’ sera reveal 41 potential biomarkers for RA that can be applied in clinical research. Chapter 3 describes another LC-MS/MS based method developed for the structural analysis of N-glycan released from the monoclonal antibody, immunoglobin G. Higher-energy collision dissociation (HCD) was the surprior technique utilized to identify glycopeptide fragments. The results show that 19 and 23 N-glycan structures were determined from standard and modified mAb samples respectively by using SimGlycan software, while 38 and 35 glycan structures were recognized by manually mapping respectively. 13 N-glycoforms, out of 26 overlapped glycan structures, were identified with significant alterations by comparing standard sample (sample A) and modified mAb (sample B) utilizing our method. In Chapter 4, we comprehensively studied hemagglutinin by using LC-MS/MS and MALDI from both proteomic perspective and glycomics prospective. After confirmed and verified protein sequence and glycosylation sites, galactose-specific quantitation was performed with exoglycosidase digestion combined HPLC with fluorescence detection. The MALDI-MS/MS based method was utilized to confirm glycan structures. The results in this dissertation provide insights into the significance of protein glycosylation alterations as RA biomarkers, and these quantitative methods can be reapplied to any other disease biomarkers screening for clinical researchers.

Proteomics refers to global characterization of the full set of proteins present in a biological sample. Various analytical disciplines contribute to proteomics but mass spectrometry became method of choice for analysis of complex protein samples. Mass spectrometry allows for high throughput analysis of the proteome but, moreover, it has the ability to acquire higher-order information such as post-translational modifications (PTM). Glycosylation is the most abundant PTM on eukaryotic proteins. This dissertation will focus on method development for structural proteomics that will be utilized to explain the glycoproteome of obligate intracellular protozoan parasite Toxoplasma gondii as a model system. Optimization of sample preparation is addressed in the first part of this dissertation. Sample preparation for mass spectrometry analysis is a critical step in the proteomics workflow because the quality and reproducibility of sample extraction and preparation significantly impacts the separation and identification capabilities of mass spectrometers. Also, there are problems unique to intracellular parasites as limited amount, host cell impurity and choice of the host. The additional obstacle is to extract only glycosylated proteins for which there is no one standard method. Here we report the optimal sample preparation method utilizing agarose bound Concanavalin A (Con A) beads to efficiently pull down glycoproteins, dialyze and analyze them using MuDPIT. This method was further enhanced by passing the non-retained protein fraction (first flow-through) through a second Con A column and then passing the second non-retained protein fraction (second flow-through) through the third Con A column (3 sequential pull-downs) yielding 394 benchmark proteins. Glycoproteome of Toxoplasma gondii is not yet fully understood. However, evidence suggests that glycosylation could be essential for cyst formation and maintenance which is characteristic of chronic stage of disease. The focus of the second part of dissertation is to better understand the differences in glycoproteomes of tachizoites and tissue cysts. Cyst proteins pulled down using optimized sample preparation method that do not appear in the tachyzoites pulldowns could be critical elements in the structural stability of the tissue cyst.

Infectious diseases caused by viruses such as influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose major threats to human health. Glycosylation, a post-translational modification critical for biological functions including receptor recognition and binding, cell adhesion, and protein folding, is a key mediator of the interaction between viruses and host cells. IAV and SARS-CoV-2 recognize and bind to glycans on host cells prior to uptake by the cells; by the same token, the glycoproteins hemagglutinin of IAV and the spike protein of SARS-CoV-2 are the targets of both host immune molecules and vaccines. The diversity of glycans, structures made up of oligosaccharide residues in complex, branched configurations, can in part be attributed to the push and pull of evolutionary pressures from infectious disease agents such as these viral pathogens. Evolving host glycans may gain the ability to evade recognition by viruses, and likewise, the evolution of viral glycans may result in viral evasion from immune responses. Thus, for a complete understanding of host-pathogen interactions, detailed characterization of glycoproteins that quantitatively measures changes in glycosylation is necessary. However, a number of factors makes quantitative characterization of glycoproteins difficult. Firstly, glycans are highly heterogeneous with dozens of possible glycans at a given glycosylation site and different occupancy levels at each site. Secondly, a particular glycoform may have very low abundance, making the signals difficult to detect. Thirdly, it is difficult to achieve deep, quantitative measurement of glycoprotein glycans using conventional liquid chromatography-mass spectrometry experiments. The usual mass spectrometry methods are not adequate because they are biased towards selecting higher abundance precursors, which leave many glycopeptide glycoforms undetected. This dissertation begins with an assessment of the current state-of-the-art of glycoproteomics using mass spectrometry to give context to our primary research discussed in subsequent chapters. Chapter 2 describes the use of a modified Tanimoto similarity coefficient to quantify the glycosylation similarity between two variants of a strain of IAV, wild-type and mutant, both expressed in embryonated chicken eggs. Our results indicate that even subtle changes in the amino acid sequence of hemagglutinin can result in measurably distinct glycosylation. Chapter 3 expands the number of comparisons of IAV strains made in the previous chapter to include strains produced in a mammalian expression vector, Madin-Darby canine kidney cells. We show that the choice of expression system can change the population of glycoforms at some but not necessarily all glycosylation sites. In addition, we explore data-independent acquisition mass spectrometry to improve upon sensitivity and selectivity of glycopeptide identification. In Chapter 4, this data-independent acquisition method is applied to the quantitative characterization of SARS-CoV-2 spike protein. The work presented here provides a significant contribution toward improving the confident detection and assignment of site-specific glycopeptides. Furthermore, understanding how to measure changes in glycosylation in related viral glycoprotein variants offers opportunities to include consideration of specific glycosylations in the design of vaccines to potentially improve efficacy against continually evolving viruses.

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing proteins and nucleic acid cargo. These vesicles are released by almost all cell types and provide an effective and ubiquitous path for intercellular communication and transmission of pathogenic and signaling molecules among cells. Research into potential biomarkers isolated from EV has been propelled by the development of methods and tools to acquire them by minimally and non-invasive means, which reinforces their great diagnostic potential. In the context of cancer, this opens the door to apply EV based liquid biopsy for early detection prior to alternate, more prevailing diagnostic tools like imaging studies. In autoimmune diseases, EVs play a crucial role in immune responses and as immunomodulatory agents as they can modulate the function of a wide variety of immune cells, especially in antigen-presenting cells (APCs). Several efforts have been made to study EVs and their cargo in numerous disease models, but very few in autoimmunity. Autoimmune diseases are chronic, have been underexplored especially in the omics area, and their diagnosis and treatment rely on traditional therapy. Therefore, there is a need for efficient methods to elucidate biomarkers that could provide additional layers of information for treatment, diagnosis, and prognosis. Additionally, protein post-translational modifications (PTMs), such as phosphorylation, glycosylation, and acetylation, are involved in multiple essential cellular processes and represent an important mechanism of regulation for cellular physiological functions, leading to the development of effective and targeted therapeutics. Discovery and profiling PTMs have established the relevance of PTMs in EVs and associated EV functions and novel applications. This dissertation proposes integrated proteomic strategies to efficiently isolate and analyze EVs in human plasma from different types of pathologies like cancer and autoimmune diseases. The main focus is the development of the platforms, to not only isolate the proteome from EVs, but also PTMs including phosphorylation, glycosylation and acetylation, simultaneously. Chapter one, which is the core of this dissertation, describes the platform to sequentially isolate and analyze the EV proteome, phosphoproteome and glycoproteome from human plasma. Chapters two and three focus on the ongoing application of this platform with slight modifications into different disease models, in this case breast cancer subtypes and autoimmune diseases.