Influence of Secondary Structure on the Fragmentation of Protonated Peptides

Abstract

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...

Fragmentation of protonated ions of peptides containing cysteine, cysteine sulfinic acid, and cysteine sulfonic acid

“…The b 3 ion (m/z 405) is also produced, but in very low abundance (Figure 4c). Tsaprailis et al [21] reported that collisional activation of protonated peptide ions containing arginine and aspartic acid or glutamic acid residues results in selective cleavages at the C-terminal side of the acidic residues if the number of ionizing protons does not exceed the number of arginine residues. Our data with the [M ϩ H] ϩ ion of the PRCGVPDVA appear not to be consistent with this view because the selective cleavage occurs at the amide bond between valine and proline, which gives the b 5 ion.…”

Structure and reactivity of an and a*n peptide fragments investigated using isotope labeling, tandem mass spectrometry, and density functional theory calculations

“…Other alternative ion structures can be derived if His, Arg, Asp, or Lys occupies the C-terminal position in b ions [23][24][25][26]. In these cases the side-chain nucleophiles of the preceding amino acids are responsible for cleavage of the amide bond or reisomerization of the primarily formed oxazolone ring.…”

Computational investigation and hydrogen/deuterium exchange of the fixed charge derivative tris(2,4,6-Trimethoxyphenyl) phosphonium: Implications for the aspartic acid cleavage mechanism

“…Statistical [19 -24] and mechanistic [25][26][27][28][29][30][31][32] analyses have led to a better understanding of peptide fragmentation behavior. Specifically, these studies have revealed residuespecific preferential cleavage N-terminal to proline (Pro), C-terminal to aspartic acid (Asp) and glutamic acid (Glu), and C-terminal to oxidized cysteine (e.g., cysteine sulfinic acid and cysteine sulfonic acid) [19,20,[33][34][35][36][37][38][39][40][41][42][43].…”