Dataset Information

Direct Monitoring of Protein O-GlcNAcylation by High-Resolution Native Mass Spectrometry.

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, CK2? 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.

SUBMITTER: Leney AC

PROVIDER: S-EPMC5565903 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Direct Monitoring of Protein O-GlcNAcylation by High-Resolution Native Mass Spectrometry.

Leney Aneika C AC Rafie Karim K van Aalten Daan M F DMF Heck Albert J R AJR

ACS chemical biology 20170628 8

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 emplo ...[more]

PMID: 28609614

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Dataset Information

Direct Monitoring of Protein O-GlcNAcylation by High-Resolution Native Mass Spectrometry.

Publications

Direct Monitoring of Protein O-GlcNAcylation by High-Resolution Native Mass Spectrometry.

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