0-GlcNAc Modification Study by In Vitro Glycosylation: A Mass Spectrometry Approach

Abstract

<p> 0-GlcNAc modification is a single N-acetylglucosamine (GlcNAc) modification on Ser or Thr residue on protein. The addition and removal of the 0GlcNAc molecule are controlled by two enzymes (OGT and NCOAT). In this study, I expressed and purified the two enzymes involved in the 0-GlcNAc modification. A method was developed for the synthesis and purification of the peptide substrate YSDSPSTST for in vitro glycosylation and characterized the OGT enzyme activity by the in vitro glycosylation and H3 labeling. A method was developed based on detection of glycosylation peptide by mass spectrometry after separation by capillary liquid chromatography (CapLC). The optimization of mass spectrometry parameters was done using synthesized standard glycopeptide YSDSPSgTST ("Sg" represents 0-GlcNAc modified Serine). The in vitro modification site was determined by CID after alkaline J3-elimination. Furthers experiment could include detection of 0-GlcNAc modification of protein substrate both in vitro and in vivo. This will give a better understanding of the dynamics of 0-GlcNAc modification. </p>