Enrichment-Free Identification of Native Definitive (EnFIND) O-glycoproteome of antibodies in autoimmune diseases

The detection of O-glycosylation at the proteome level has long been a challenging task and a roadblock for O-linked protein glycosylation research. We report an Enrichment-Free Identification of Native Definitive (EnFIND) O-glycoproteome using Trapped Ion Mobility Spectrometry coupled to TOF Mass Spectrometry (TIMS-TOF MS) for direct analysis of protein O-glycosylation in native samples with minimum sample requirement. This approach enabled separation of O-glycopeptide isomers, resolution of O-glycosites and O-glycoform, reduction of sample complexity, and increased sensitivity, thus greatly enhancing analysis of the O-glycoproteome of cell lysates, human serum and exosomes. In addition, we found that antibodies in human serum are highly O-glycosylated on variable, especially hypervariable regions and constant regions, which significantly increases antibody diversity. This method was used to successfully identify characteristic O-glycosylation features of autoimmune diseases.