Mapping O-glycosylation Sites Using OpeRATOR and LC-MS

Methods Mol Biol. 2021:2271:155-167. doi: 10.1007/978-1-0716-1241-5_11.

Abstract

O-glycosylation is a difficult posttranslational modification to analyze. O-glycans are labile and often cluster making their analysis by LC-MS very challenging. OpeRATOR is an O-glycan specific protease that cleaves the protein backbone N-terminally of glycosylated serine and threonine residues. This enables the generation of glycopeptides of suitable size for mapping O-glycosylation sites in detail by bottom-up LC-MS analysis. In this chapter we demonstrate a simple workflow for in-depth analysis of O-glycosylation sites on heavily glycosylated proteins using OpeRATOR digestion and HILIC-MS/MS analysis.

Keywords: Glycopeptides; Glycoproteomics; LC-MS; Mass spectrometry; O-glycan specific protease; O-glycosylation; OpeRATOR.

MeSH terms

  • Chromatography, Liquid*
  • Complement C1 Inhibitor Protein / analysis*
  • Glycosylation
  • Peptide Hydrolases / metabolism*
  • Peptide Mapping*
  • Protein Processing, Post-Translational*
  • Proteolysis
  • Research Design
  • Spectrometry, Mass, Electrospray Ionization*
  • Tandem Mass Spectrometry*
  • Workflow

Substances

  • Complement C1 Inhibitor Protein
  • SERPING1 protein, human
  • Peptide Hydrolases