LAUSR

Changes in mucin-type O-glycosylation of human proteins affect protein function, immune response, and cancer progression. As O-glycoproteins are characterized by microheterogeneity of diverse O-glycans with no conserved sequence and macroheterogeneity of multiple glycosylation sites on serine and/or threonine in human proteins, the assessment of different mucin-types, such as Tn-antigen, core 1, and core 2, and their extended core types in O-glycopeptides is extremely challenging. Here, we present an O-GlycoProteome Analyzer (O-GPA) that automatically classifies mucin-type O-glycosylation using higher-energy collisional dissociation (HCD) in mass spectrometry. First, we estimated the number of GlcNAc residues using the intensity ratio of GlcNAc-specific fragment ions (HexNAc-CH6O3 and HexNAc-2H2O) over GalNAc-specific fragment ions (HexNAc-C2H6O3 and HexNAc-C2H4O2) in the HCD spectrum. Further, we classified the different mucin-types of O-glycopeptides from characteristic B2 (HexNAc2) or Y2α (PEP+HexNAc2), and Y2β (PEP+HexNAcHex) fragment ions along with the number of GlcNAc. Furthermore, O-GPA automatically determined single or multiple O-glycosylation regardless of mucin-types. The mucin-type of O-glycopeptides from human urine and plasma was confirmed with an overall accuracy of 96%. We found 97 core 1, 56 core 2, 13 extended core 1, and 12 extended core 2 of glycopeptides from urine; and 22 core 1, 13 core 2, seven extended core 1, one extended core 2, and one Tn-antigen from plasma. Our strategy can be used to successfully characterize specific mucin-types of O-glycoproteins in human biological samples.