The influence glutamic acid in protonated b₃ → b₂ formation from VGEIG and related analogs

A direct pathway for the fragmentation of peptide b₃ fragment ions to b₂ ions has, until now, not been identified. Experimental evidence for the formation of a b₃ anhydride structure and isomerization to an extended macrocycle is demonstrated here by comparison of the completely different fragmentation patterns of the b₃ ions generated from protonated VGEIG and its methyl ester. In particular, the absence of a b₂ ion in the fragmentation spectrum of the methyl ester b₃ indicates that facile fragmentation of an anhydride-type b₃ is responsible for virtually all b₂ ions formed. The stability of this b₃ structure and the ease with which it fragments to the b₂ may be responsible for the relatively high abundance of the b₃ and b₂ ions. IRMPD action spectroscopy measurements indicate the presence of a ring protonated oxazolone in the b₂ population. VGEIG and three related analogs, VA_LEIG, VA_DEIG, and V(Aib)EIG were studied by QCID-HDX-SORI experiments in an FT-ICR instrument, and provide significant evidence for extensive alpha proton scrambling in an ion-molecule complex formed between the b₂ and neutral loss fragment following formation of the b₂. MS³ and HDX of VG(2,2-d2)EIG indicate that the scrambled b₂ ions have the same structure as the unscrambled b₂. Based on these data and with the support of molecular modeling, we propose a new mechanism for this scrambling, in which the alpha protons are transferred in a multistep pathway during an ion-molecule complex formed between the b₂ and amino-terminated anhydride ring neutral loss component.