期刊
ACS CHEMICAL BIOLOGY
卷 12, 期 8, 页码 2078-2084出版社
AMER CHEMICAL SOC
DOI: 10.1021/acschembio.7b00371
关键词
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资金
- Wellcome Trust Investigator Award [110061]
- Roadmap Initiative Proteins Work, - Netherlands Organization for Scientific Research (NWO) [184.032.201]
- MSMed program, - European Union's Horizon Framework Programme [686547]
- Biotechnology and Biological Sciences Research Council [1416998] Funding Source: researchfish
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, CK2a 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.
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