Fragment Correlation Mass Spectrometry Enables Direct Characterization of Disulfide Bond Cleavage Pathways of Therapeutic Peptides

Therapeutic peptides that are connected by disulfide bonds are often difficult to analyze by traditional tandem mass spectrometry without chemical modification. Using fragment correlation mass spectrometry, we analyzed 56 pairs of fragment ions generated from an equimolar (10 μM) mixture of three cyclic peptides, achieving sequence coverage of 86%, 100%, and 75% for octreotide, desmopressin, and the structural analogue of desmopressin, respectively. In all detected fragment ion pairs, only 20% of the fragment ions are terminal ions, with most of the measured ions only detected by fragment correlation mass spectrometry. From the peak volumes in the covariance map, we calculated branching ratios of each disulfide bond fragmentation pathway, providing a direct measurement of the probability of each fragmentation without requiring alteration of the chemical structure of the analytes.