Advancing Ultraviolet Photodissociation Mass Spectrometry for Precision Mapping of Protein Glycosylation

PROJECT SUMMARY

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 β-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.