Loss of Internal Backbone Carbonyls: Additional Evidence for Sequence-Scrambling in Collision-Induced Dissociation of y-Type Ions

It is shown that y-type ions, after losing C-terminal H₂O or NH₃, can lose an internal backbone carbonyl (CO) from different peptide positions and yield structurally different product fragment ions upon collision-induced dissociation (CID). Such CO losses from internal peptide backbones of y-fragment ions are not unique to a single peptide and were observed in four of five model peptides studied herein. Experimental details on examples of CO losses from y-type fragment ions for an isotopically labeled AAAAHAA-NH₂ heptapeptide and des-acetylated-α-melanocyte-stimulating hormone (dα-MSH) (SYSMEHFRWGKPV-NH₂) are reported. Results from isotope labeling, tandem mass spectrometry (MSⁿ), and ion mobility-mass spectrometry (IM-MS) confirm that CO losses from different amino acids of m/z-isolated y-type ions yield structurally different ions. It is shown that losses of internal backbone carbonyls (as CID products of m/z-isolated y-type ions) are among intermediate steps towards formation of rearranged or permutated product fragment ions. Possible mechanisms for generation of the observed sequence-scrambled a-“like” ions, as intermediates in sequence-scrambling pathways of y-type ions, are proposed and discussed. ?