Site-Specific O-Glycosylation Analysis by Liquid Chromatography-Mass Spectrometry with Electron-Transfer/Higher-Energy Collisional Dissociation.

O-glycosylation is a major post-translational modification of proteins. Accurate and detailed analysis to reveal O-glycosylation patterns at each site (site-specific O-glycosylation analysis) is essential to deeply understand glycoprotein function. Recent reports also demonstrated that unintended O-glycosylation occurs on therapeutic fusion glycoproteins; therefore, it is increasingly important to perform detailed and exhaustive O-glycosylation analysis during the development of therapeutic glycoproteins. Here, we describe a method of in-depth site-specific O-glycosylation analysis by liquid chromatography-mass spectrometry using electron-transfer/higher-energy collisional dissociation (EThcD) and database analysis.