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

Algorithms for Glycan Structure Identification with Tandem Mass Spectrometry

Glycosylation is a frequently observed post-translational modification (PTM) of proteins. It has been estimated over half of eukaryotic proteins in nature are glycoproteins. Glycoprotein analysis plays a vital role in drug preparation. Thus, characterization of glycans that are linked to proteins has become necessary in glycoproteomics. Mass spectrometry has become an effective analytical technique for glycoproteomics analysis because of its high throughput and sensitivity. The large amount of spectral data collected in a mass spectrometry experiment makes manual interpretation impossible and requires effective computational approaches for automated analysis. Different algorithmic solutions have been proposed to address the challenges in glycoproteomics analysis based on mass spectrometry. However, new algorithms that can identify intact glycopeptides are still demanded to improve result accuracy. In this research, a glycan is represented as a rooted unordered labelled tree and we focus on developing effective algorithms to determine glycan structures from tandem mass spectra. Interpreting the tandem mass spectra of glycopeptides with a de novo sequencing method is essential to identifying novel glycan structures. Thus, we mathematically formulated the glycan de novo sequencing problem and propose a heuristic algorithm for glycan de novo sequencing from HCD tandem mass spectra of glycopeptides. Characterizing glycans from MS/MS with a de novo sequencing method requires high-quality mass spectra for accurate results. The database search method usually has the ability to obtain more reliable results since it has the assistance of glycan structural information. Thus, we propose a de novo sequencing assisted database search method, GlycoNovoDB, for mass spectra interpretation

Identification of Glycopeptides with Multiple Hydroxylysine O-Glycosylation Sites by Tandem Mass Spectrometry

Glycosylation is one of the most common post-translational modifications in proteins, existing in ∼50% of mammalian proteins. Several research groups have demonstrated that mass spectrometry is an efficient technique for glycopeptide identification; however, this problem is still challenging because of the enormous diversity of glycan structures and the microheterogeneity of glycans. In addition, a glycopeptide may contain multiple glycosylation sites, making the problem complex. Current software tools often fail to identify glycopeptides with multiple glycosylation sites, and hence we present GlycoMID, a graph-based spectral alignment algorithm that can identify glycopeptides with multiple hydroxylysine O-glycosylation sites by tandem mass spectra. GlycoMID was tested on mass spectrometry data sets of the bovine collagen α-(II) chain protein, and experimental results showed that it identified more glycopeptide-spectrum matches than other existing tools, including many glycopeptides with two glycosylation sites

Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics

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

The analysis of alphaâ 1â antitrypsin glycosylation with direct LCâ MS/MS

A liquid chromatographyâ tandem mass spectrometry (LCâ MS/MS)â based methodology has been developed to differentiate coreâ and antennaryâ fucosylated glycosylation of glycopeptides. Both the glycosylation sites (heterogeneity) and multiple possible glycan occupancy at each site (microheterogeneity) can be resolved via intact glycopeptide analysis. The serum glycoprotein alphaâ 1â antitrypsin (A1AT) which contains both coreâ and antennaryâ fucosylated glycosites was used in this study. Sialidase was used to remove the sialic acids in order to simplify the glycosylation microheterogeneity and to enhance the MS signal of glycopeptides with similar glycan structures. β1â 3,4 galactosidase was used to differentiate coreâ and antennaryâ fucosylation. Inâ source dissociation was found to severely affect the identification and quantification of glycopeptides with low abundance glycan modification. The settings of the mass spectrometer were therefore optimized to minimize the inâ source dissociation. A threeâ step mass spectrometry fragmentation strategy was used for glycopeptide identification, facilitated by pGlyco software annotation and manual checking. The collision energy used for initial glycopeptide fragmentation was found to be crucial for improved detection of oxonium ions and better selection of Y1 ion (peptide+GlcNAc). Structural assignments revealed that all three glycosylation sites of A1AT glycopeptides contain complex Nâ glycan structures: site Asn70 contains biantennary glycans without fucosylation; site Asn107 contains biâ , triâ and tetraâ antennary glycans with both coreâ and antennaryâ fucosylation; site Asn271 contains biâ and triâ antennary glycans with both coreâ and antennaryâ fucosylation. The relative intensity of coreâ and antennaryâ fucosylation on Asn107 was similar to that of the A1AT protein indicating that the glycosylation level of Asn107 is much larger than the other two sites.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146302/1/elps6432_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146302/2/elps6432.pd

Comprehensive Glycoproteomics and Glycomics Study of N-Linked Glycans and N-Glycoproteins

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

Mass Spectrometry Based Analysis of Protein N-Glycosylation in Biomarker Discovery and Gene Therapy with the Study Models-Hemophilia A Inhibitor Development and rAAV

Protein glycosylation is one of the critical post-translational modifications (PTMs) and practically engaged with a wide range of physiological and biological processes. Glycosylation is the most dynamic post-translational modification and an individual\u27s glycome changes overcome the genetic factors and get affected by environmental factors which eventually reflect his lifestyle, physiological conditions and wellbeing. The flow study, we attempted to add this information to comprehend the glycoprotein biomarker identified with inhibitor advancement and connected the glycosylation related changes to the biochemical pathway of inhibitor development against rFVIII in HA population. We performed the study with mice and human models. Plasma and IgG N-glycome examination is one of the important methodologies to identify the biomarker related to numerous conditions. The N-glycome pattern also varies in response to the treatment. The treatment-related modifications also reaffirm the observations noted in the progression of the disease. Similarly, the glycosylation can be a useful strategy to modify the protein-based drugs to enhance its mode of action. The variant of AAV can be a potential capsid engineering technique to alter the tropism and improve the gene delivery range of host cell range for engineering a better gene delivery system. The small amount of and glycan variants are difficult to detect in a complex biological mixture, which may require various enrichment strategies, and sample preparations help to enhance the detection sensitivity in mass spectrometry. Due to the with the development of state-of-the-art mass spectrometry (MS) technology, we tried to identify N-glycan biomarkers related to inhibitor development in HA. Also, we decided to study the response of the patient after emicizumab. Additionally, we identified N-glycosylation in rAAV-8, which can be a potential direction for future capsid engineering

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

Applications of ion mobility spectrometry, collision-induced dissociation and electron activated dissociation tandem mass spectrometry to structural analysis of proteins, glycoproteins and glycans

This dissertation mainly focuses on analytical method development for characterization of proteins, glycoproteins and glycans using the recently developed ion mobility spectrometry (IMS) techniques and various electron activated dissociation (ExD) tandem mass spectrometry methods. IMS and ExD have become important techniques in structure analysis of biomolecules. IMS is a gas-phase separation method orthogonal to liquid chromatography (LC) fractionation. ExD is capable of producing a large number of structurally informative fragment ions for elucidation of structural details, complementary to collision-induced dissociation (CID). We first applied the selected accumulation-trapped IMS (SA-TIMS)-electronic excitation dissociation (EED) method to analyze various mixtures of glycan isomers. Glycan linkage isomers with linear or branched structure were successfully separated and subsequently identified. Theoretical modeling was also performed to gain a better understanding of isomer separation. The calculated collisional cross section (CCS) values match well with the experimentally measured ones, and suggested that the choice of metal charge carrier and charge state is critical for successful IMS separation of isomeric glycans. In addition, a SA-TIMS-electron capture dissociation (ECD) approach was employed to study gas-phase protein conformation, as the ECD fragmentation pattern is influenced by both the charge distribution and the presence of various non-covalent interactions. We demonstrated that different conformations of protein ions in a single charge state could produce distinct fragmentation pattern, presumably because of their differences in tertiary structures and/or proton locations. The second part describes characterization of glycoproteins using LC-hot ECD. To improve the cleavage coverage of glycopeptides, hot ECD, a fragmentation method utilizing the irradiation of high-energy electrons, was optimized for both middle-down and bottom-up analyses of glycopeptides, including peptides with multiple glycosylation sites. Hot ECD was shown to be an effective fragmentation technique for sequencing of glycopeptides, even for ions in lower charge states. In addition, the online LC-hot ECD approach was applied to characterize extensively modified glycoproteins from biological sources in which all glycosylation sites could be unambiguously determined. This study expands the applications of IMS, CID and ExD to structural analysis of various biomolecules, and explores the analytical potential of combining them for investigation of complex biological systems, in particular, enzyme mechanisms