Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry

One of the major factors governing the "top-down" sequence analysis of intact multiply protonated proteins by tandem mass spectrometry is the effect of the precursor ion charge state on the formation of product ions. To more fully understand this effect, electrospray ionization coupled to a quadrupole ion trap mass spectrometer, collision-induced dissociation, and gas-phase ion/ion reactions have been employed to examine the fragmentation of the [M + 12H]12+ to [M + H]+ ions of bovine ubiquitin. At low charge states (+1 to +6), loss of NH3 or H2O from the protonated precursor and directed cleavage at aspartic acid residues was observed. At intermediate charge states, (+7, +8, and +9), extensive nonspecific fragmentation of the protein backbone was observed, with 50% sequence coverage obtained from the [M + 8H]8+ ion alone. At high charge states, (+10, +11, +12), the single dominant channel that was observed was the preferential fragmentation of a single proline residue. These data can be readily explained in terms of the current model for intramolecular proton mobilization, that is, the "mobile proton model", the mechanisms for amide bond dissociation developed for protonated peptides, as well as the structures of the multiply charged ions of ubiquitin in the gas phase, examined by ion mobility and hydrogen/deuterium exchange measurements.     

Collision-induced dissociation of intact duplex and single-stranded siRNA anions

A tandem mass spectrometry approach is demonstrated for complete sequencing of a model small interfering RNA (siRNA) based on ion trap collisional activation of intact single-stranded anions. Various charge states of the siRNA duplex and the individual strands were generated by nanoelectrospray (nano-ESI). The siRNA duplex anions were predominantly dissociated into the sense and antisense strands by collisional activation. The characteristic fragment ions (c/y- and a-B/w-ion series) from both strands were observed when higher activation amplitude was applied and when beam-type collisional activation was examined; however, the coexistence of fragment ions from both strands complicated spectral interpretation. The effect of precursor ion charge state on the dissociation of the individual sense and antisense strand siRNA anions was studied using ion trap collision-induced dissociation under various activation amplitudes. Through the activation of relatively low charge state precursor ions at relatively low excitation energy, selective backbone dissociation predominantly via the c/y channels was achieved. By applying relatively high excitation energy, the a-B/w channels also became prominent; however, the increase in spectral complexity made complete peak assignment difficult. In order to simplify the product ion spectra, proton-transfer reactions were applied, and complete sequencing of each strand was achieved. The application of tandem mass spectrometry to intact single-stranded anions demonstrated in this study can be adapted for the rapid identification of other noncoding RNAs in RNomics studies.     

Identification of bacteriophage MS2 coat protein from E. coli lysates via ion trap collisional activation of intact protein ions

Collisional activation of the intact MS2 viral capsid protein with subsequent ion/ion reactions has been used to identify the presence of this virus in E. coli lysates. Tandem ion trap mass spectrometry experiments on the +7, +8, and +9 charge states, followed by ion/ion reactions, provided the necessary sequence tag information (and molecular weight data) needed for protein identification via database searching. The most directly informative structural information is obtained from those charge states that produce a series of product ions arising from fragmentation at adjacent residues. The formation of these product ions via dissociation at adjacent amino acid residues depends greatly on the charge state of the parent ion. Database searching of the charge-state-specific sequence tags was performed by two different search engines: the ProteinInfo program from the Protein information Retrieval On-line World Wide Web Lab or PROWL and the TagIdent program from the ExPASy molecular biology server. These search engines were used in conjunction with the sequence tag information generated via collisional activation of the intact viral coat protein. These programs were used to evaluate the feasibility of generating sequence tags from collisional activation of intact multiply charged protein ions in a quadrupole ion trap.     

Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors

Electron-transfer dissociation (ETD) delivers the unique attributes of electron capture dissociation to mass spectrometers that utilize radio frequency trapping-type devices (e.g., quadrupole ion traps). The method has generated significant interest because of its compatibility with chromatography and its ability to: (1) preserve traditionally labile post-translational modifications (PTMs) and (2) randomly cleave the backbone bonds of highly charged peptide and protein precursor ions. ETD, however, has shown limited applicability to doubly protonated peptide precursors, [M + 2H]2+, the charge and type of peptide most frequently encountered in "bottom-up" proteomics. Here we describe a supplemental collisional activation (CAD) method that targets the nondissociated (intact) electron-transfer (ET) product species ([M + 2H]+*) to improve ETD efficiency for doubly protonated peptides (ETcaD). A systematic study of supplementary activation conditions revealed that low-energy CAD of the ET product population leads to the near-exclusive generation of c- and z-type fragment ions with relatively high efficiency (77 +/- 8%). Compared to those formed directly via ETD, the fragment ions were found to comprise increased relative amounts of the odd-electron c-type ions (c+*) and the even-electron z-type ions (z+). A large-scale analysis of 755 doubly charged tryptic peptides was conducted to compare the method (ETcaD) to ion trap CAD and ETD. ETcaD produced a median sequence coverage of 89%-a significant improvement over ETD (63%) and ion trap CAD (77%).     

Ion trap collisional activation of the (M + 2H)2+ - (M + 17H)17+ ions of human hemoglobin beta-chain

The parent ions of human hemoglobin beta-chain ranging in charge from 2+ to 17+ have been subjected to ion trap collisional activation. The highest charge-state ions (17+ to 13+) yielded series of products arising from dissociation of adjacent residues. The intermediate charge-state ions (12+ to 5+) tended to fragment preferentially at the N-terminal sides of proline residues and the C-terminal sides of acidic residues. Many, but not all, of the possible cleavages at proline, aspartic acid, and glutamic acid residues were represented in the spectra. The lowest charge-state ions were difficult to dissociate with high efficiency and yielded spectra with poorly defined product ion signals. This observation is attributed to sequential fragmentations arising from losses of small molecules such as water and/or ammonia. The poor fragmentation efficiency observed for the low charge states is due at least in part to the low trapping wells used to store the ions. Higher ion stabilities due to lower Coulombic repulsion and charges being sequestered at highly basic sites may also play an important role. Ion/ion proton-transfer reactions involving protein parent ions allows for the formation of a wide range of parent ion charge states. In addition, the ion/ion proton-transfer reactions involving protein dissociation products simplify interpretation of the product ion spectra.     

Activation of intact electron-transfer products of polypeptides and proteins in cation transmission mode ion/ion reactions

Cationic peptide electron-transfer products that do not fragment spontaneously are exposed to ion trap collisional activation immediately upon formation while they pass through a high-pressure collision cell (Q2), where the electron-transfer reagent anions are stored. Radial ion acceleration, which is normal to the ion flow, is implemented by applying an auxiliary dipolar alternating current to a pair of opposing rods of the Q2 quadrupole array at a frequency in resonance with the surviving electron-transfer products. Collisional cooling of cations in the pressurized Q2 ensures efficient overlap of the positive and negative ions for ion/ion reactions and also gives rise to relatively long residence times (milliseconds) for ions in Q2, making it possible to fragment ions via radial excitation during their axial transmission. The radial activation for transmission mode electron-transfer ion/ion reactions has been demonstrated with a doubly protonated tryptic peptide, a triply protonated phosphopeptide, and [M + 7H]7+ ions of ubiquitin. In all cases, significant increases in fragment ion yields and structural information from electron-transfer dissociation (ETD) were observed, suggesting the utility of this method for improving transmission mode ETD performance for relatively low charge states of peptides and proteins.     

Electron-induced dissociation of singly charged organic cations as a tool for structural characterization of pharmaceutical type molecules

Collision-induced dissociation (CID) and electron-induced dissociation (EID) have been investigated for a selection of small, singly charged organic molecules of pharmaceutical interest. Comparison of these techniques has shown that EID carried out on an FTICR MS and CID performed on a linear ion trap MS produce complementary data. In a study of 33 molecule-cations, EID generated over 300 product ions compared to 190 product ions by CID with an average of only 3 product ions per precursor ion common to both tandem MS techniques. Even multiple stages of CID failed to generate many of the product ions observed following EID. The charge carrying species is also shown to have a very significant effect on the degree of fragmentation and types of product ion resulting from EID. Protonated species behave much like the ammonium adduct with suggestion of a hydrogen atom from the charge carrying species strongly affecting the fragmentation mechanism. Sodium and potassium are retained by nearly every product ion formed from [M + Na](+) or [M + K](+) and provide information to complement the EID of [M + H](+) or [M + NH(4)](+). In summary, EID is proven to be a fitting partner to CID in the structural elucidation of small singly charged ions and by studying EID of a molecule-ion holding different charge carrying species, an even greater depth of detail can be obtained for functional groups commonly used in synthetic chemistry.     

Phosphorylation site identification via ion trap tandem mass spectrometry of whole protein and peptide ions: bovine alpha-crystallin A chain

Tandem mass spectrometry was applied both to ions of a tryptic fragment and intact protein of bovine alpha-crystallin A chain to localize the single site of phosphorylation. The [M + 19H](19+) to [M + 11H](11+) charge states of both phosphorylated and unphosphorylated bovine alpha-crystallin A chain whole protein ions were subjected to collisional activation in a quadrupole ion trap. Ion parking was used to increase the number of parent ions over that yielded by electrospray. Ion-ion proton-transfer reactions were used to reduce the product ion charge states largely to +1 to simplify spectral interpretation. In agreement with previous studies on whole protein ion fragmentation, both protein forms showed backbone cleavages C-terminal to aspartic acid residues at lower charge states. The phosphorylated protein showed competitive fragmentation between backbone cleavage and the neutral loss of phosphoric acid. Analysis of which backbone cleavage products did or did not contain the phosphate was used to localize the site of phosphorylation to one of two possible serine residues. A tryptic digest of the bovine alpha-crystallin A chain yielded a phosphopeptide containing one missed cleavage site. The peptide provided information complementary to that obtained from the intact protein and localized the modified serine to residue 122. Fragmentation of the triply charged phosphopeptide yielded five possible serine phosphorylation sites. Fragmentation of the doubly charged phosphopeptide, formed by ion/ion proton-transfer reactions, positively identified the phosphorylation site as serine-122.     

Charge determination of product ions formed from collision-induced dissociation of multiply protonated molecules via ion/molecule reactions.

The use of ion/molecule reactions involving multiply protonated ions derived from electrospray for the determination of the charges of product ions formed from collision-induced dissociation is described. The experiments are carried out with a quadrupole ion trap capable of multiple stages of mass spectrometry. The approach is illustrated with proton transfer from a product ion from quadruply protonated melittin, and from a product ion from the (M + 20H)20+ ion from horse myoglobin, to 1,6-diaminohexane. The major product ion from quadruply protonated bovine insulin is used to illustrate the use of a clustering reaction with 1,6-diaminohexane. The ion trap is shown to be a particularly useful tool for employing both collisional activation and low-energy ion/molecule reactions in the same experiment to determine product ion charge.     

ESI-MS/MS for the differentiation of diastereomeric pyrimidine glycols in mononucleosides

Pyrimidine glycols, or 5,6-dihydroxy-5,6-dihydropyrimidines, are primary lesions in DNA induced by reactive oxygen species. In this article, we report the preparation and tandem mass spectrometry (MS/MS) characterization of the two cis diastereomers of the glycol lesions of 2'-deoxyuridine, 5-methyl-2'-deoxycytidine, and thymidine. Our results show that collisional activation of the [M + Na]+ ions of all the three pairs of cis isomers and that of the [M + H]+ ions of the 2'-deoxyuridine glycols and 5-methyl-2'-deoxycytidine glycols give a facile loss of a water molecule. Interestingly, the water loss occurs more readily for the 6S isomer than for the 6R isomer. Likewise, product ion spectra of the [M - H]- ions of the two cis isomers of the 2'-deoxyuridine glycols and thymidine glycols show more facile loss of water for the 6S isomer than for the 6R isomer. MS/MS acquired at different collisional energies gave similar results, which establishes the reproducibility of spectra.     

Automated tandem mass spectrometry by orthogonal acceleration TOF data acquisition and simultaneous magnet scanning for the characterization of petroleum mixtures

The automated acquisition of the product ion spectra of all precursor ions in a selected mass range by using a magnetic sector/orthogonal acceleration time-of-flight (oa-TOF) tandem mass spectrometer for the characterization of complex petroleum mixtures is reported. Product ion spectra are obtained by rapid oa-TOF data acquisition and simultaneous scanning of the magnet. An analog signal generator is used for the scanning of the magnet. Slow magnet scanning rates permit the accurate profiling of precursor ion peaks and the acquisition of product ion spectra for all isobaric ion species. The ability of the instrument to perform both high- and low-energy collisional activation experiments provides access to a large number of dissociation pathways useful for the characterization of precursor ions. Examples are given that illustrate the capability of the method for the characterization of representative petroleum mixtures. The structural information obtained by the automated MS/MS experiment is used in combination with high-resolution accurate mass measurement results to characterize unknown components in a polar extract of a refinery product. The exhaustive mapping of all precursor ions in representative naphtha and middle-distillate fractions is presented. Sets of isobaric ion species are separated and their structures are identified by interpretation from first principles or by comparison with standard 70-eV EI libraries of spectra. The utility of the method increases with the complexity of the samples.     

Tandem mass spectrometry of ribonuclease A and B: N-linked glycosylation site analysis of whole protein ions

Recently, an approach for the "top down" sequence analysis of whole protein ions has been developed, employing electrospray ionization, collision-induced dissociation, and ion/ion proton-transfer reactions in a quadrupole ion trap mass spectrometer. This approach has now been extended to an analysis of the [M + 12H]12+ to [M + 5H]5+ ions of ribonuclease A and its N-linked glycosylated analogue, ribonuclease B, to determine the influence of the posttranslational modification on protein fragmentation. In agreement with previous studies on the fragmentation of a range of protein ions, facile gas-phase fragmentation was observed to occur along the protein backbone at the C-terminal of aspartic acid residues, and at the N-terminal of proline, depending on the precursor ion charge state. Interestingly, no evidence was found for gas-phase deglycosylation of the N-linked sugar in ribonuclease B, presumably due to effective competition from the facile amide bond cleavage channels that "protect" the N-linked glycosidic bond from cleavage. Thus, localization of the posttranslational modification site may be determined by analysis of the "protein fragment ion mass fingerprint".     

Effects of charge state and cationizing agent on the electron capture dissociation of a peptide

Electron capture dissociation (ECD) is a promising method for de novo sequencing proteins and peptides and for locating the positions of labile posttranslational modifications and binding sites of noncovalently bound species. We report the ECD of a synthetic peptide containing 10 alanine residues and 6 lysine residues uniformly distributed across the sequence. ECD of the (M + 2H)(2+) produces a limited range of c (c(7)-c(15)) and z (z(9)-z(15)) fragment ions, but ECD of higher charge states produces a wider range of c (c(2)-c(15)) and z (z(2)-z(6), z(9)-z(15)) ions. Fragmentation efficiency increases with increasing precursor charge state, and efficiencies up to 88% are achieved. Heating the (M + 2H)(2+) to 150 degrees C does not increase the observed range of ECD fragment ions, indicating that the limited products are due to backbone cleavages occurring near charges and not due to effects of tertiary structure. ECD of the (M + 2Li)(2+) and (M + 2Cs)(2+) produces di- and monometalated analogues of the same c and z ions observed from the (M + 2H)(2+), with the abundance of dimetalated fragment ions increasing with fragment ion mass, a result consistent with the metal cations being located near the peptide termini to minimize Coulombic repulsion. In stark contrast to the ECD results, collisional activation of cesiated dications overwhelmingly results in ejection of Cs(+). The abundance of cesiated fragment ions formed from ECD of the (M + Cs + Li)(2+) exceeds that of lithiated fragment ions by 10:1. ECD of the (M + H + Li)(2+) results in exclusively lithiated c and z ions, indicating an overwhelming preference for neutralization and cleavage at protonated sites over metalated sites. These results are consistent with preferential neutralization of the cation with the highest recombination energy.     

A quadrupole ion trap mass spectrometer with three independent ion sources for the study of gas-phase ion/ion reactions

An instrument for the study of gas-phase ion/ion reactions in which three independent sources of ions, namely, two electrospray ionization sources and one atmospheric sampling glow discharge ionization source, are interfaced to a quadrupole ion trap mass analyzer is described. This instrument expands the scope of gas-phase ion/ion reaction studies by allowing for manipulation of the charge states of multiply charged reactant and product ions. Examples are provided involving the formation of protein-protein complexes in the gas phase. Complexes with charge states that cannot be formed from reactant ion charge states present in the normal electrospray charge state distributions can be formed in the new apparatus. Strategies that rely on both reactant ion charge state manipulation and product ion charge state manipulation are demonstrated. In addition, simplification of product ion spectra generated from dissociation of complexes formed via ion/ion reactions can be effected by using the discharge source to reduce the charge state of the product ions to primarily 1+.     

Identification of microcystin toxins from a strain of Microcystis aeruginosa by liquid chromatography introduction into a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer

The cyclic heptapeptide microcystin toxins produced by a strain of Microcystis aeruginosa that has not been investigated previously were separated by liquid chromatography and identified by high-accuracy m/z measurements of their [M + H]+ ions and the fragment ions produced by collision-activated dissociation of the [M + H]+ ions. The cyanobacteria B2666 strain was cultured in a standard growth medium, and the toxins were released from the cells, extracted from the aqueous phase, and concentrated using standard procedures. The microcystins were separated by reversed-phase microbore liquid chromatography and introduced directly into a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer with electrospray ionization. The known microcystins (MC) MC-LR, MC-LA, [MeSer7]MC-LR, MC-LL, MC-LF, and MC-L(Aba) were identified along with the two previously unreported structural variants [Asp3]MC-LA and [Asp3]MC-LL. In addition to the [M + H]+ ions, accurate m/z measurements were made of 12-18 product ions for each identified microcystin. The mean difference between measured and calculated exact m/z was less than 2 parts per million, which often allowed assignment of unique compositions to the observed ions. A mechanism is presented that accounts for an important collision-activated dissociation process that gives valuable sequence ions from microcystins that do not contain arginine. The analytical technique used in this work is capable of supporting fairly rapid and very reliable identifications of known microcystins when standards are not available and of most structural variants independent of additional information from other analytical techniques.     

Collisional activation with random noise in ion trap mass spectrometry.

Random noise applied to the end caps of a quadrupole ion trap is shown to be an effective means for the collisional activation of trapped ions independent of mass/charge ratio and number of ions. This technique is compared and contrasted with conventional single-frequency collisional activation for the molecular ion of N,N-dimethylaniline, protonated cocaine, the molecular anion of 2,4,6-trinitrotoluene, and doubly pronated neuromedin U-8. Collisional activation with noise tends to produce more extensive fragmentation than the conventional approach due to the fact that product ions are also kinetically excited in the noise experiment. The efficiency of the noise experiment in producing detectable product ions relative to the conventional approach ranges from being equivalent to being a factor of 3 less efficient. Furthermore, discrimination against low mass/charge product ions is apparent in the data from multiply charged biomolecules. Nevertheless, collisional activation with random noise provides a very simple means for overcoming problems associated with the dependence of single-frequency collisional activation on mass/charge ratio and the number of ions in the ion trap.     

A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry

Recent advances in phosphopeptide enrichment prior to mass spectrometric analysis show genuine promise for characterization of phosphoproteomes. Tandem mass spectrometry of phosphopeptide ions, using collision-activated dissociation (CAD), often produces product ions dominated by the neutral loss of phosphoric acid. Here we describe a novel method, termed Pseudo MS(n), for phosphopeptide ion dissociation in quadrupole ion trap mass spectrometers. The method induces collisional activation of product ions, those resulting from neutral loss(es) of phosphoric acid, following activation of the precursor ion. Thus, the principal neutral loss product ions are converted into a variety of structurally informative species. Since product ions from both the original precursor activation and all subsequent neutral loss product activations are simultaneously stored, the method generates a "composite" spectrum containing fragments derived from multiple precursors. In comparison to analysis by conventional MS/MS (CAD), Pseudo MS(n) shows improved phosphopeptide ion dissociation for 7 out of 10 synthetic phosphopeptides, as judged by an automated search algorithm (TurboSEQUEST). A similar overall improvement was observed upon application of Pseudo MS(n) to peptides generated by enzymatic digestion of a single phosphoprotein. Finally, when applied to a complex phosphopeptide mixture, several phosphopeptides mis-assigned by TurboSEQUEST under the conventional CAD approach were successfully identified after analysis by Pseudo MS(n).     

Mass spectrometry and tandem mass spectrometry of citrus limonoids

Methods for atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) of citrus limonoid aglycones and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of limonoid glucosides are reported. The fragmentation patterns of four citrus limonoid aglycones (limonin, nomilin, obacunone, and deacetylnomilin) and six limonoid glucosides, that is, limonin 17-beta-D-glucopyranoside (LG), nomilin 17-beta-D-glucopyranoside (NG), nomilinic acid 17-beta-D-glucopyranoside (NAG), deacetyl nomilinic acid 17-beta-D-glucopyranoside (DNAG), obacunone 17-beta-D-glucopyranoside (OG), and obacunoic acid 17-beta-D-glucopyranoside (OAG) were investigated using a quadruple mass spectrometer in low-energy collisionally activated dissociation (CAD). The four limonoid aglycones and four limonoid glucosides (LG, OG, NAG, and DNAG) were purified from citrus seeds; the other two limonoid glucosides (NG and OAG) were tentatively identified in the crude extract of grapefruit seeds by ESI mass spectrometry in both positive and negative ion analysis. Ammonium hydroxide or acetic acid was added to the mobile phase to facilitate ionization. During positive ion APCI analysis of limonoid aglycones, protonated molecular ion, [M + H]+, or adduct ion, [M + NH3 + H]-, was formed as base peaks when ammonium hydroxide was added to the mobile phase. Molecular anions or adduct ions with acetic acid ([M + HOAc - H] and [M + HOAc]-) or a deprotonated molecular ion were produced during negative ion APCI analysis of limonoid aglycones, depending on the mobile-phase modifier used. Positive ion ESI-MS of limonoid glucosides produced adduct ions of [M + H + NH3]+, [M + Na]+, and [M + K]+ when ammonium hydroxide was added to the mobile phase. After collisionally activated dissociation (CAD) of the limonoid aglycone molecular ions in negative ion APCI analysis, fragment ions indicated structural information of the precursor ions, showing the presence of methyl, carboxyl, and oxygenated ring structure. CAD of the adduct ion [M + H + NH3]+ of limonoid glucosides produced the aglycone moiety corresponding to each glucoside. The combination of mass spectrometry and tandem mass spectrometry provides a powerful technique for identification and characterization of citrus limonoids.     

Ion trap versus low-energy beam-type collision-induced dissociation of protonated ubiquitin ions

The beam-type and ion trap collision-induced dissociation (CID) behaviors of protonated bovine ubiquitin ions were studied for charge states ranging from +6 to +12 on a modified triple quadrupole/linear ion trap tandem mass spectrometer. Both beam-type CID and ion trap CID were conducted in a high-pressure linear ion trap, followed by proton-transfer ion/ion reactions to reduce the charge states of product ions mostly to +1. The product ions observed under each activation condition were predominantly b- and y-type ions. Fragmentation patterns showed a much stronger dependence on parent ion charge state with ion trap CID than with beam-type CID using nitrogen as the collision gas, with preferential cleavages C-terminal to aspartic acid at relatively low charge states, nonspecific fragmentation at moderate charge states, and favored cleavages N-terminal to proline residues at high charge states. In the beam-type CID case, extensive cleavage along the protein backbone was noted, which yielded richer sequence information (77% of backbone amide bond cleavages) than did ion trap CID (52% of backbone amide bond cleavages). Collision gas identity and collision energy were also evaluated in terms of their effects on the beam-type CID spectrum. The use of helium as collision gas, as opposed to nitrogen, resulted in CID behavior that was sensitive to changes in collision energy. At low collision energies, the beam-type CID data resembled the ion trap CID data with preferential cleavages predominant, while at high collision energies, nonspecific fragmentation was observed with increased contributions from sequential fragmentation.     

Charge-remote fragmentation of odd-electron peptide ions

Comparison between the gas-phase fragmentation of odd-electron M+*, [M + H]2+*, and [M - 2H]-* ions of model peptides suggests that charge-remote radical-driven fragmentation pathways play an important role in the dissociation of odd-electron peptide ions. We have found that charge-remote processes are responsible for a variety of side-chain losses from the precursor ion and some backbone fragmentation. These fragmentation pathways most likely involve hydrogen abstraction by the radical site that initiates subsequent cleavages. These findings are generally relevant to our understanding of the fragmentation patterns of odd-electron peptide ions produced through various approaches including the capture of low-energy electrons, electron detachment, and electron transfer.