Glyco-DIA: a method for quantitative O-glycoproteomics with in silico-boosted glycopeptide libraries

We report a LC-MS/MS O-glycoproteomics strategy using Data Independent Acquisition (DIA) mode that holds the potential for enabling direct analysis of O-glycoproteins with characterization of sites and structures of O-glycans on a proteome-wide scale with quantification of stoichiometries. To explore the use of a DIA strategy for O-glycoproteomics, we built a spectral library of O-glycopeptides with the most common core1 O-glycan structures. This Glyco-DIA library consists of sublibraries obtained from cell lines and human serum, and it currently covers 2,076 O-glycoproteins (11,452 unique glycopeptide sequences) and the five most common core1 O-glycan structures. Applying the Glyco-DIA library to human serum without enrichment for glycopeptides enabled us to identify and quantify nearly 293 distinct glycopeptide sequences bearing up to 5 different core1 O-glycans from 159 glycoproteins in a singleshot analysis. The DIA method is expandable and widely applicable to different glycoproteomes, and it may represent the first direct and comprehensive approach to glycoproteomics.