Glycosylation plays a structural and functional role in all fundamental features of proteins in cells. Despite substantial evidence suggesting O-glycosylation is vital in cellular processes, the nature of O-linked glycans and the specific locations of O-glycans are not well characterized. To a large extent, the consensus sequence motif N-X-S/T enables reliable prediction of N-glycosylation sites, but our knowledge of O-glycosylation is hampered by a lack of simple consensus motifs. As a result, our current understanding of the impact of O-glycosylation is incomplete. A major obstacle to comprehensive characterization of O-glycosylation by mass spectrometry-based proteomic workflows is the prominent neutral loss of labile O-linked glycans using conventional collisional activated dissociation, hampering precise localization. Moreover, no global enzyme exists that can cleave every possible O-glycan ?-O-glycosidic linkage, thwarting enzymatic glycomic analysis. Mass spectrometry coupled with ultraviolet photodissociation (UVPD) is positioned to be an important tool in glycoproteomics by enabling residue-level resolution of PTMs of proteins implicated in human health. UVPD can be harnessed to provide simultaneously high sequence coverage of peptides and retention of labile modifications, thus allowing O-glycan mapping and structural characterization. My development of innovative LC-UVPD-MS strategies suitable for both targeted and global glycoproteomic applications will provide new insight into the correlation of glycosylation with disease pathways and drive new biological questions.
The attachment of sugars to proteins, a process called glycosylation, is an important pathway often implicated in numerous health-related cellular mechanisms that mediate the immune response, developmental defects, and cancer. This proposal focuses on the development of innovative tandem mass spectrometry methodologies to map glycosylation sites of proteins and allow more detailed insight into structure/function relationships of proteins implicated in disease.
