The influence of acid−base interactions on the gas-phase dissociation of a series of protonated peptides was investigated. Peptides containing both acidic residues [aspartic (D), glutamic (E), and cysteic acid (C*)] and basic residues [arginine (R)] were dissociated by different activation methods that allow different time frames for dissociation. The synthetic peptides investigated differ systematically in the number and position of arginine residue(s) and include R LDIFSDF R , R LEIFSEF R , R LDIFSDF, LDIFSDF R , LEIFSEF R , LDIFSDF, R LCIFSCF R , R LAIFSCF R , R LCIFSAF R , R LC*IFSC*F R , R LAIFSC*F R , and R LC*IFSAF R (where C* denotes cysteic acid). It was observed that the number of ionizing protons relative to the number of basic residues in peptides containing acidic residues is a contributing factor in the fragmentation behavior. Nonselective cleavages along the peptide backbone occur when the number of ionizing protons exceeds the number of arginine residues, while dominant cleavages adjacent to the acidic residues predominate when the number of ionizing protons equals the number of arginine residues. In particular, enhanced b7/y2, and y6, y2 singly charged fragment ions were detected for the doubly protonated R LDIFSDF R and singly protonated LDIFSDF R precursor ions, respectively. These are the result of enhanced cleavage of the DF bond in the doubly protonated R LDIFSDF R and the DI plus DF bonds in the singly protonated LDIFSDF R . Abundant d and b-H2SO3 product ions indicative of specific cleavages adjacent to C* were observed in the cysteic acid-containing peptides when the number of ionizing protons equaled the number of arginine residues. Dominant cleavages at glutamic acid(s) were also observed for doubly protonated R LEIFSEF R and singly protonated LEIFSEF R when longer dissociation times were available. Preferential cleavage(s) at the acidic residue(s) occurs on the microsecond time scale for aspartic acid and greater than microsecond time scale for glutamic acid. This different behavior for aspartic vs glutamic acid is likely to have important implications in mass spectrometry-based sequencing strategies. However, the product ion spectra of most of the peptides investigated ( R LDIFSDF R , R LDIFSDF, LDIFSDF R , LEIFSEF R , and LDIFSDF) were found to be very similar under the array of activation methods used. These included surface-induced dissociation in a quadrupole tandem mass spectrometer, high-energy collision-induced dissociation in a hybrid sector/time-of flight mass spectrometer, and sustained off-resonance irradiation in a Fourier transform mass spectrometer. The unique fragmentation of peptides containing basic and acidic residues is rationalized as evidence for the existence of gas-phase intramolecular solvation that strongly influences their fragmentation. We propose that it is the available acidic proton(s) on the acidic residue(s) not involved in solvating the protonated arginine that is initiating the dominant cleavage(s). Electrospray ionization/SID frag...

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(+)CH(2)C(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, b5(2+) and b6(2+) 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 RVYIHPF. Based on all of the results, a selective fragmentation mechanism for enhanced cleavage at histidine involving an atypical b ion structure is proposed.

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