Diagonal Chromatography for the Isolation of O -Linked β-N-acetyl-D-Glucosaminylated ( O -GlcNAcylated) Peptides in HeLa Cell Extracts

• DOI: 10.1080/00032719.2024.2333335
• Publication Date: 2024-04-01T15:04:15Z
• Authors: Pongsakorn Thawor...; Churat Weeraphan; Supinya Thanapong...; Charinrat Saechan; Worrawit Wanichsuwan; Kanitta Srinoun; Natta Tansila; Chris Verathamjamras; Voraratt Champatt...; Daranee Chokchaic...; Chantragan Srisomsap; Jisnuson Svasti; Hansuk Buncherd
• Keywords: HeLa; Isolation

Protein O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a dynamic post-translational modification process that plays an essential role in biological activities. Growing evidence indicates aberration of O-GlcNAcylation associated with various diseases, e.g. diabetes, neurological and cancers. However, the mechanistic studies are lagging behind other modifications due to its extremely low abundance, limited analytical tools, specificity. Herein, diagonal strong cation exchange chromatography was applied enrich O-GlcNAc glycosylated peptides prior mass spectrometric analysis by liquid chromatography/ion trap tandem spectrometry (LC–MS/MS). In this strategy, O-GlcNAcylated were first enzymatically labeled azide-modified galactosamine (GalNAz) fractionated (SCX) chromatography. Tris(carboxyethyl)phosphine (TCEP) reduces azido group GalNAz-modified primary amine group. TCEP-induced reduction separated from unmodified SCX. By reversed-phase LC–MS/MS secondary SCX fractions, isolated identified mixtures O-GlcNAc-modified HeLa cell extract. A total 250 sites on 215 proteins identified. Therefore, novel method could be potential tool for isolation site peptides.