학술논문
Diagonal Chromatography for the Isolation of O -Linked β-N-acetyl-D-Glucosaminylated (O -GlcNAcylated) Peptides in HeLa Cell Extracts.
Document Type
Article
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ISSN
0003-2719
Abstract
AbstractProtein 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. [ABSTRACT FROM AUTHOR]