Investigation of gas phase ion structure for proline-containing b(2) ion.

Unusual fragmentation was observed for doubly charged VPDPR in which cleavage C-terminal to proline and N-terminal to aspartic acid yielded b(2) (+ a(2))/y(3) complementary ions. This unique fragmentation is contradictory to trends previously established by statistical analysis of peptide tandem mass (MS/MS) spectra. Substitution of alanine for aspartic acid (i.e., VPAPR) did not change the fragmentation, indicating the cleavage was not directed by aspartic acid. Fragmentation patterns for VPAPR and V(NmA)APR (NmA = N-methyl alanine) were compared to determine whether conformational constraints from proline's cyclic side-chain contribute to b(2) ion formation. While both peptide sequences fragmented to yield b(2)/y(3) ions, only VPAPR produced a(2) ions, suggesting the VP b(2) ion is structurally different from the V(NmA) b(2) ion. Instead, the V(NmA) b(2) ion was accompanied by an ion corresponding to formal loss of 71. The suspected structural differences were confirmed by isolation and fragmentation of the respective b(2) ions (i.e., MS(3) spectra). Evidence supporting a diketopiperazine structure for the VP b(2) ion is reported. Fragmentation patterns for the VP b(2) ion and a synthetic VP diketopiperazine showed great similarity. N-terminal acetylation of VPAPR prevented the formation of the VP b(2) ion, presumably by blocking nucleophilic attack by the N-terminal amine on the carbonyl oxygen of the protonation site. Acetylation of the N-terminus for V(NmA)APR did not prevent the formation of the V(NmA) b(2) ion, indicating the V(NmA) b(2) ion has a structure, presumably that of an oxazolone, which requires no attack by the N-terminus for formation. Finally, high-resolution, accurate mass measurements determined that the V(NmA) (b(2)-71) ion results from losing a portion of valine from oxazolone V(NmA) b(2) ion, rather than cross-ring cleavage of the alternate diketopiperazine.