Thawornpan P.Weeraphan C.Thanapongpichat S.Saechan C.Wanichsuwan W.Srinoun K.Tansila N.Verathamjamras C.Champattanachai V.Chokchaichamnankit D.Srisomsap C.Svasti J.Buncherd H.Mahidol University2024-04-142024-04-142024-01-01Analytical Letters (2024)00032719https://repository.li.mahidol.ac.th/handle/123456789/97964Protein O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a dynamic post-translational modification process that plays an essential role in biological activities. Growing evidence indicates that aberration of O-GlcNAcylation is associated with various diseases, e.g. diabetes, neurological diseases, and cancers. However, the mechanistic studies of O-GlcNAcylation are lagging behind other post-translational modifications due to its extremely low abundance, limited analytical tools, and specificity. Herein, diagonal strong cation exchange chromatography was applied to enrich the O-GlcNAc glycosylated peptides prior to mass spectrometric analysis by liquid chromatography/ion trap tandem mass spectrometry (LC–MS/MS). In this strategy, the O-GlcNAcylated peptides were first enzymatically labeled with an azide-modified galactosamine (GalNAz) and fractionated by strong cation exchange (SCX) chromatography. Tris(carboxyethyl)phosphine (TCEP) reduces the azido group in GalNAz-modified peptides to a primary amine group. TCEP-induced reduction of GalNAz-modified peptides was separated from unmodified peptides by diagonal SCX. By reversed-phase LC–MS/MS analysis of secondary SCX fractions, O-GlcNAcylated peptides were isolated and identified from the mixtures of O-GlcNAc-modified and unmodified peptides in HeLa cell extract. A total of 250 O-GlcNAcylation sites on 215 proteins were identified. Therefore, this novel method could be a potential tool for the isolation and site analysis of O-GlcNAc-modified peptides.ChemistryBiochemistry, Genetics and Molecular BiologyMedicineArticleSCOPUS10.1080/00032719.2024.23333352-s2.0-851897903451532236X