Fragment Correlation\nMass Spectrometry Enables Direct\nCharacterization of Disulfide Bond Cleavage Pathways of Therapeutic\nPeptides

Therapeutic\npeptides that are connected by disulfide bonds are\noften difficult to analyze by traditional tandem mass spectrometry\nwithout chemical modification. Using fragment correlation mass spectrometry,\nwe analyzed 56 pairs of fragment ions generated from an equimolar\n(10 μM) mixture of three cyclic peptides, achieving sequence\ncoverage of 86%, 100%, and 75% for octreotide, desmopressin, and the\nstructural analogue of desmopressin, respectively. In all detected\nfragment ion pairs, only 20% of the fragment ions are terminal ions,\nwith most of the measured ions only detected by fragment correlation\nmass spectrometry. From the peak volumes in the covariance map, we\ncalculated branching ratios of each disulfide bond fragmentation pathway,\nproviding a direct measurement of the probability of each fragmentation\nwithout requiring alteration of the chemical structure of the analytes.