Cell surface proteome characterization using cell -binding arrays and mass spectrometry

Cell surface proteins are embedded in or associated with a plasma membrane that provides a physical barrier between cells and the extracellular environment. An important characteristic of the cell surface proteome is that it contains receptor proteins which bind to extracellular ligands to initiate cellular responses such as changes in gene expression. Another pivotal feature of cell surface proteins is that most of them are glycosylated, which is essential to cell recognition, adhesion, and cell-cell interactions. Moreover, these cell surface receptor expression and glycosylation patterns vary depending on the cell state or type. Thus, characterization of these cell surface proteins is essential to understand basic cellular functions.;In this thesis, cell-binding arrays were employed to explore cell surface receptor expression and cell surface carbohydrate expression patterns without isolation or purification of the cell surface proteins. These arrays utilize aldehyde-terminated self-assembled monolayers (SAMs) on gold surfaces to react with amine-bearing biomolecules. Protein ligands or lectins were attached to the SAMs through their lysine residues and then captured whole cells through a ligand-receptor or a lectin-glycoprotein interaction. Characterization of vascular endothelial growth factor (VEGF) receptors involved in angiogenesis and fibroblast growth factor (FGF) receptors responsible for human embryonic stem cell self-renewal was made using these cell-binding arrays.;Since the glycosylation patterns of cell surface proteins display a structural diversity which depends on cell type, the cell surface glycoproteins are good candidate surface markers to distinguish the different cell types. In this thesis, lectin arrays were utilized to explore cell surface carbohydrate expression patterns of endothelial and fibroblast cells, and novel candidate cell surface markers to differentiate the two cell types were identified using mass spectrometry.;Finally, tyrosine-phosphorylated proteins and proteins that interact with them, which play critical roles in maintaining stem cell self-renewal, were enriched using phosphotyrosine antibodies and analyzed using mass spectrometry.