Relative energetics of fragmentation of protonated peptides are investigated by using electrospray ionization/ surface-induced dissociation (ESI/SID) tandem mass spectrometry. ESI/SID fragmentation efficiency curves (percent fragmentation versus laboratory collision energy) are presented for 20 oligopeptides and are a measure of how easily a peptide fragments. The relative positions of the ESI/SID fragmentation efficiency curves depend on several parameters which include peptide composition (e.g., presence/absence of a basic amino acid residue) and peptide size. The ESI/SID fragmentation efficiency curves, in combination with quantum chemical calculations, provide a unique approach to substantiate and refine the mobile proton model for peptide fragmentation. Selected peptides are also investigated to further test and confirm the mobile proton model; these include doubly-protonated peptides and chemically-modified peptides (i.e., acetylated and fixed-charge derivatized peptides). Doubly-protonated peptides fragment more easily than the singly-protonated forms of the same peptides, with a sequence dependence for the difference in energy required for the fragmentation of singly-vs doubly-protonated peptides. Acetylation at the amino terminus and arginine side chain leads to a decrease in basicity and a corresponding lower energy onset for fragmentation than for the unmodified form of the peptide. Fixing the site of charge by addition of trimethylammonium acetyl to the amino terminus, i.e., eliminating the mobile proton, results in a higher energy onset than that for the protonated form of the same peptide. Curves for doubly protonated peptides with two adjacent basic residues (Arg, Arg) suggest the localization of the two protons at the two basic side chains rather than at opposite termini of the peptide.

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...

Collision-induced dissociation (CID) is a common ion activation technique used to energize massselected peptide ions during tandem mass spectrometry. Characteristic fragment ions form from the cleavage of amide bonds within a peptide undergoing CID, allowing the inference of its amino acid sequence. The statistical characterization of these fragment ions is essential for improving peptide identification algorithms and for understanding the complex reactions taking place during CID. An examination of 1465 ion trap spectra from doubly charged tryptic peptides reveals several trends important to understanding this fragmentation process. While less abundant than y ions, b ions are present in sufficient numbers to aid sequencing algorithms. Fragment ions exhibit a characteristic series-specific relationship between their masses and intensities. Each residue influences fragmentation at adjacent amide bonds, with Pro quantifiably enhancing cleavage at its N-terminal amide bond and His increasing the formation of b ions at its C-terminal amide bond. Fragment ions corresponding to a formal loss of ammonia appear preferentially in peptides containing Gln and Asn. These trends are partially responsible for the complexity of peptide tandem mass spectra.Tandem mass spectrometry (MS/MS) of peptides is a central technology for proteomics, enabling the identification of thousands of peptides from a complex mixture. [1][2][3][4] This increasingly widespread technique relies upon the fragmentation of peptides by collisioninduced dissociation (CID), but the chemistry behind the fragmentation process is complex and not comprehensively understood. [5][6][7][8] Peptides undergo CID after they are isolated from other ions by their mass-to-charge (m/z) ratios. Peptides in an acidic solution are introduced to the vacuum of the mass spectrometer via electrospray ionization. 9 The peptide ions are accelerated during CID, leading to more energetic collisions with the ion trap's inert gas molecules. The mobile proton model 10 describes how the added internal energy causes the ionizing proton(s) on each peptide to transfer intramolecularly until one destabilizes a peptide bond, resulting in the cleavage of that bond and the production of two fragments. While more energetic techniques may cleave many classes of bonds within the peptide structure, low-energy CID preferentially breaks the amide bonds. Once the fragment ions are produced, the mass spectrometer records their m/z ratios in a tandem mass spectrum.Determining the sequence of a peptide from its tandem spectrum is complicated by the variety and variability of the fragment ions produced. Cleavage of amide bonds results in b and y Figure 1). b ions may fragment further to produce a ions. 13 If only these three ions were produced for every amide bond in a 10-residue peptide, the fragment ion spectrum would contain 27 peaks. This ideal spectrum differs from experimental spectra as a result of several causes. First, a subset of the expected fragment ions may not be present. Second...

Data mining was performed on 28 330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chemical interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx-Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissociation patterns for singly charged Arg- and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides, a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from singly charged peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s) in multiply protonated peptides. Enhanced cleavages C-terminal to branched aliphatic residues (Ile, Val, Leu) are observed in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages N-terminal to these residues are observed in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns observed expand our knowledge of peptide gas-phase fragmentation behaviors and may be useful in algorithm development that employs improved models to predict fragment ion intensities.