A Mechanistic Investigation of the Enhanced Cleavage at Histidine in the Gas-Phase Dissociation of Protonated Peptides

Enhanced gas-phase cleavage of peptides adjacent to histidine was investigated. The peptides examined were angiotensins III (RVYIHPF) and IV (VYIHPF) as well as synthetic peptide analogues with altered key residues ((R)VYI-X-Z-F; X = F or H and Z = A, P, or Sar) or a fixed charge M3P ⁺CH₂C(O)-VYIHPF. While all singly protonated peptide ions containing both histidine and arginine fragment nonselectively, the doubly protonated peptide ions with arginine and histidine, and the singly protonated peptides containing histidine but not arginine, cleave in a selective manner. In particular, dominant complementary b⁺/y⁺ product ions resulting from cleavage between the HP amide bond are observed. For the fixed-charge derivative, selective cleavage occurs only if a proton is added to produce a doubly charged precursor. The results are consistent with involvement of a protonated histidine in the selective cleavage. The ratio of b ⁺/y⁺ is determined by the identity of the residue C-terminal to histidine and by the ability of protonated histidine to transfer a proton to the C-terminal leaving fragment. This was probed further by systematically changing the residue C-terminal to histidine and by alkylating histidine. The results indicate that while b⁺/y⁺ complementary ion pairs dominate in doubly protonated RVYIHPF, b ₅²⁺ and b₆²⁺ product ions dominate the spectra of doubly protonated RVYIHAF. Also, dominant b₅ ²⁺ product ions are observed when the histidine side chain is alkylated (H*) in doubly protonated RVYIH*PF. Based on all of the results, a selective fragmentation mechanism for enhanced cleavage at histidine involving an atypical b ion structure is proposed.