Classification of Mucin-Type -Glycopeptides Using Higher-Energy Collisional Dissociation in Mass Spectrometry

Changes in mucin-type -glycosylation of human proteins affect protein function, immune response, and cancer progression. Since -glycoproteins are characterized by the microheterogeneity of diverse -glycans with no conserved sequence and the 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 -glycopeptides, is extremely challenging. Here, we present an (-GPA) that automatically classifies mucin-type -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-CHO and HexNAc-2HO) over GalNAc-specific fragment ions (HexNAc-CHO and HexNAc-CHO) in the HCD spectrum. Furthermore, we classified the different mucin types of -glycopeptides from characteristic B (HexNAc) or Y (PEP + HexNAc), and Y (PEP + HexNAcHex) fragment ions, along with the number of GlcNAc. Furthermore, -GPA automatically determined single or multiple -glycosylation, regardless of the mucin types. The mucin type of -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 glycopeptides from urine; and 22 core 1, 13 core 2, 7 extended core 1, 1 extended core 2, and 1 Tn-antigen from plasma. Our strategy can be used to successfully characterize specific mucin types of -glycoproteins in human biological samples.