Influence of secondary structure on the fragmentation of protonated peptides

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 RLDIFSDFR, RLEIFSEFR, RLDIFSDF, LDIFSDFR, LEIFSEFR, LDIFSDF, RLCIFSCFR, RLAIFSCFR, RLCIFSAFR, RLC*IFSC*FR, RLAIFSC*FR, and RLC*IFSAFR (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 b(7)/y(2), and y(6), y(2) singly charged fragment ions were detected for the doubly protonated RLDIFSDFR and singly protonated LDIFSDFR precursor ions, respectively. These are the result of enhanced cleavage of the DF bond in the doubly protonated RLDIFSDFR and the DI plus DF bonds in the singly protonated LDIFSDFR. 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 RLEIFSEFR and singly protonated LEIFSEFR 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 (RLDIFSDFR, RLDIFSDF, LDIFSDFR, LEIFSEFR, 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 Right 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 fragmentation efficiency curves (percent fragmentation versus laboratory collision energy) are also presented for these peptides. The positions of the curves for the doubly protonated, double arginine-containing peptides (RLDIFSDFR, RLEIFSEFR) relative to those for the doubly protonated but single arginine-containing peptides (LDIFSDFR, RLDIFSDF) are consistent with localization of charge at the two R side chains in the former peptides and formation of a heterogeneous population of protonated peptides in the latter peptides. These curve positions and the nonselective fragmentation in the peptide devoid of arginine residues (LDIFSDF) are consistent with the mobile proton model, which relates ease of fragmentation to ease of nonselective intramolecular proton transfer within the protonated peptides.