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Leney AC, Rafie K, van Aalten DMF, Heck AJR. Direct Monitoring of Protein O-GlcNAcylation by High-Resolution Native Mass Spectrometry. ACS chemical biology 2017 12(8) 28609614
Abstract:
O-GlcNAcylation is one of the most abundant metazoan nuclear-cytoplasmic post-translational modifications. Proteins modified by O-GlcNAc play key cellular roles in signaling, transcription, metabolism, and cell division. Mechanistic studies on protein O-GlcNAcylation are hampered by the lack of methods that can simultaneously quantify O-GlcNAcylation, determine its stoichiometry, and monitor O-GlcNAcylation kinetics. Here, we demonstrate that high-resolution native mass spectrometry can be employed to monitor the small mass shifts induced by modification by O-GlcNAc on two known protein substrates, CK2α and TAB1, without the need for radioactive labeling or chemoenzymatic tagging using large mass tags. Limited proteolysis enabled further localization of the O-GlcNAc sites. In peptide-centric MS analysis, the O-GlcNAc moiety is known to be easily lost. In contrast, we demonstrate that the O-GlcNAc is retained under native MS conditions, enabling precise quantitative analysis of stoichiometry and O-GlcNAcylation kinetics. Together, the data highlight that high resolution native MS may provide an alternative tool to monitor kinetics on one of the most labile of protein post-translational modifications, in an efficient, reliable, and quantitative manner.
O-GlcNAc proteins:
CSK21, TAB1
Species: Homo sapiens
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Borodkin VS, Schimpl M, Gundogdu M, Rafie K, Dorfmueller HC, Robinson DA, van Aalten DM. Bisubstrate UDP-peptide conjugates as human O-GlcNAc transferase inhibitors. The Biochemical journal 2014 457(3) 24256146
Abstract:
Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, eliminating the GlcNAc pyranoside ring. Goblin1 co-crystallizes with OGT, revealing an ordered C₃ linker and retained substrate-binding modes, and binds the enzyme with micromolar affinity, inhibiting glycosyltransfer on to protein and peptide substrates.
O-GlcNAc proteins:
TAB1
Species: Homo sapiens
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