Characteristic Fragmentation Behavior of Linear and Cyclic O-Linked Glycopeptide and Their Peptide Skeletons in MALDI-TOF/TOF MS

O-linked glycopeptides have attracted attention because of their ability to control peptide conformation and function via glycosylation. Cyclic peptides have also attracted attention for their various physiological and physical activities, owing to the conformational constraints associated with cyclization. We found that cyclic antifreeze glycopeptides showed characteristic peptide chain fragmentation patterns in the MALDI-TOF/TOF MS analysis. The fragmentation patterns were characterized by the parallel occurrence of side-chain fragmentation of threonine residues and fragmentation within the macrocyclic peptide bond associated with ring opening. Furthermore, the tendency for side chain fragmentation was altered depending on the adduct ions, the presence or absence of glycans, and the substitution of threonine with serine. These fragmentation mechanisms have been suggested to be influenced by conformational changes in cyclic peptides, along with adduct ion coordination or structural modifications, particularly threonine residues. This finding is expected to contribute to the prediction of peptide conformation based on fragmentation tendencies and to the search for physiologically active O-linked glycopeptides and cyclic (glyco)peptides.