5 years ago

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

Direct Monitoring of Protein O-GlcNAcylation by High-Resolution Native Mass Spectrometry
Daan M. F. van Aalten, Albert J. R. Heck, Aneika C. Leney, Karim Rafie
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.

Publisher URL: http://dx.doi.org/10.1021/acschembio.7b00371

DOI: 10.1021/acschembio.7b00371

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