Title:
MS-Based Chemical Proteomics Studies of Extracellular Glycoproteins: Identification, Quantification, and Dynamics

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Suttapitugsakul, Suttipong
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Wu, Ronghu
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Abstract
Extracellular proteins, including cell-surface and secreted proteins, play crucial roles in cell-cell communication, cell-matrix interactions, and the regulation of most extracellular biological processes. The majority of these proteins, if not all, are glycosylated, which affects their functions, properties, stability, and dynamics. Despite their importance, surface glycoproteins cannot be easily studied due to their low abundance, glycan heterogeneity, and surface specificity, surface glycoproteins cannot be easily studied. This thesis work is focused on the study of extracellular glycoproteins, including surface and secreted glycoproteins, through mass spectrometry (MS)-based proteomics. First, an enrichment method based on metabolic labeling with a sugar analog, copper-free click chemistry, and MS-based proteomics is employed to target cell-surface glycoproteins from multiple cell lines. The method is universal for all glycan types, targets surface glycoproteins only on the extracellular side, works under physiological conditions, and reveals site-specific glycosylation information. Both the relative abundance across the cells and the absolute abundance within the cells are quantified to generate a useful resource for surface glycoprotein distribution. The approach to study surface glycoproteins is then combined with multiplexed proteomics quantification using the tandem mass tag (TMT) reagents to determine the dynamics of cell-surface glycoproteins in the innate immune response to bacterial infection. The dynamics of surface glycoproteins on monocytes and macrophages are determined and compared, and the monocyte-to-macrophage differentiation is demonstrated as a source of the different responses in the two cell types. The dynamics of surface glycoproteins in cells during the epithelial-to-mesenchymal transition (EMT) is then investigated, and proteins that participate in the partial EMT state are uncovered. For secreted glycoproteins, especially in the cell culture model, the analysis is often not comprehensive due to the presence of serum proteins in the media required for cell growth and proliferation. Therefore, a method to study secreted glycoproteins in the cell culture model without serum depletion is developed by combining the enrichment method for surface glycoproteins, benefitting from the synthesis of extracellular glycoproteins through the classical secretory pathway, with a signal boosting technique through multiplexed proteomics quantification. The potential biological sources of secreted glycoproteins to use as a boosting sample are first determined, and the importance of the boosting-to-sample ratio is also demonstrated. This approach quantifies hundreds of secreted glycoproteins, including cytokines, from monocytes and macrophages in the innate immune response to bacterial infection model and from Hep G2 cells during the EMT process. Additionally, the reduction and alkylation steps of the bottom-up proteomics workflow are optimized to maximize the number of proteins and peptides that can be identified by MS. An enrichment step with thiopropyl Sepharose 6B beads for cysteine-containing peptides is further investigated. Other post-translational modifications are also explored, including phosphorylation in neuroblastoma tumors to understand their response to chemoimmunotherapeutic treatment. Not only the methods developed from this thesis will be useful for the study of extracellular glycoproteins, but the information regarding cell-surface and secreted glycoproteins is also unprecedented and will lead to a better understanding of their functions, benefiting the biological and biomedical fields.
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2021-03-24
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