Influence of cysteine to cysteic acid oxidation on the collision-activated decomposition of protonated peptides: Evidence for intraionic interactions

Oxidation of cysteine residues to cysteic acids in C-terminal arginine-eontaining peptides (such as those derived by tryptic digestion of proteins) strongly promotes the formation of multiple members of the Y? series of fragment ions following low energy collision-activated decomposition (CAD) of the protonated peptides, Removal of the arginine residue abolishes the effect, which is also attenuated by conversion of the arginine to dimethylpyrim-idylornithine. The data indicate the importance of an intraionic interaction between the cysteic acid and arginine side-chains. Low energy CAD of peptides which include cysteic acid and histidine residues, also provides evidence for intraionic interactions. It is proposed that these findings are consistent with the general hypothesis that an increased heterogeneity (with respect to location of charge) of the protonated peptide precursor ion population is beneficial to the generation of a high yield of product ions via several charge-directed, low energy fragmentation pathways. Furthermore, these data emphasize the significance of gas-phase conformations of protonated peptides in determining fragmentation pathways.     

Charge promotion of low-energy fragmentations of peptide ions.

We have examined the hypothesis that structural features which predispose to localization of charge at a strongly favored site are not conducive to the low-energy fragmentation of peptide ions via a multiplicity of pathways. Consistent with this proposal, it is demonstrated that the formation of N- or C-terminal pre-charged derivatives is detrimental to the formation of sequence-specific product ions following low-energy collisional activation. Protonation of pre-charged derivatives (yielding doubly charged ions) restores favorable fragmentation properties; the effect is attributed to the fragmentation-directing properties of the proton which may occupy one of several sites. Similarly, a doubly protonated peptide which incorporates a C-terminal arginine residue as a single strongly favored site of protonation exhibits favored low-energy fragmentations attributable to location of the second proton at one of several sites remote from the C-terminus.     

Charge location directs electron capture dissociation of peptide dications

The effect of peptide dication charge location on electron capture dissociation (ECD) fragmentation pattern is investigated. ECD fragmentation patterns are compared for peptides with amide and free acid C-terminal groups. ECD of free acid compared with C-terminally amidated peptides with basic residues near the N-terminus demonstrates increased formation of a-type ions. Similarly, ECD of free acid compared with C-terminally amidated peptides with basic residues near the C-terminus exhibits increased formation of y-type ions. Alteration of the peptide sequence to inhibit the formation of charged side chains (i.e., amino acid substitution and acetylation) provides further evidence for charge location effect on ECD. We propose that formation of zwitterionic peptide structures increases the likelihood of amide nitrogen protonation (versus basic side chains), which is responsible for the increase in a- and y-type ion formation.     

精氨酸残基在质子化多肽RRMKWKK的气相碰撞诱导解离过程中的作用

用ESI/MS-MS方法研究了质子化多肽RRMKWKK 在低能气相碰撞诱导解离(CID)条件下的碰撞能和解离路径. 研究结果表明, [M+2H]2+和[M+3H]3+的CID断裂曲线和断裂位点相似. 但质子化多肽所含正电荷个数不同时, 产生同一碎片离子的初始碰撞能不同. 碱性氨基酸残基精氨酸(Arg)的支链是多肽RRMKWKK质子化时质子优先结合的位点, 导致含有Arg的多肽在气相碰撞诱导解离条件下解离时需要较高的碰撞能. 在用质谱方法研究含精氨酸残基的多肽时应选择质子个数比多肽中Arg个数多1个的母体离子. 质子化多肽RRMKWKK的结构AM1计算结果表明, 质子化RRMKWKK中两个相邻精氨酸在空间上相互分离, 库伦斥力的影响不足以改变质子的优先结合位点.     

Primary and secondary locations of charge sites in angiotensin II (M + 2H)2+ ions formed by electrospray ionization

High-energy tandem mass spectrometry and molecular dynamics calculations are used to determine the locations of charge in metastably decomposing (M + 2H)2+ ions of human angiotensin II. Charge-separation reactions provide critical information regarding charge sites in multiple charged ions. The most probable kinetic energy released (Tm.p.) from these decompositions are obtained using kinetic energy release distributions (KERDs) in conjunction with MS/MS (MS2), MS/MS/MS (MS3), and MS/MS/MS/MS (MS4) experiments. The most abundant singly and doubly charged product ions arise from precursor ion structures in which one proton is located on the arginine (Arg) side chain and the other proton is located on a distal peptide backbone carbonyl oxygen. The MS3 KERD experiments show unequivocally that neither the N-terminal amine nor the aspartic acid (Asp) side chain are sites of protonation. In the gas phase, protonation of the less basic peptide backbone instead of the more proximal and basic histidine (His) side chain is favored as a result of reduced coulomb repulsion between the two charge sites. The singly and doubly charged product ions of lesser abundance arise from precursor ion structures in which one proton is located on the Arg side chain and the other on the His side chain. This is demonstrated in the MS3 and MS4 mass-analyzed ion kinetic energy spectrometry experiments. Interestingly, (b7" + OH)2+ product ions, like the (M + 2H)2+ ions of angiotensin II, are observed to have at least two different decomposing structures in which charge sites have a primary and secondary location.     

Improving fragmentation of poorly fragmenting peptides and phosphopeptides during collision-induced dissociation by malondialdehyde modification of arginine residues

Despite significant technological and methodological advancements in peptide sequencing by mass spectrometry, analyzing peptides that exhibit only poor fragmentation upon collision-induced dissociation (CID) remains a challenge. A major cause for unfavorable fragmentation is insufficient proton 'mobility' due to charge localization at strongly basic sites, in particular, the guanidine group of arginine. We have recently demonstrated that the conversion of the guanidine group of the arginine side chain by malondialdehyde (MDA) is a convenient tool to reduce the basicity of arginine residues and can have beneficial effects for peptide fragmentation. In the present work, we have focused on peptides that typically yield incomplete sequence information in CID-MS/MS experiments. Energy-resolved tandem MS experiments were carried out on angiotensins and arginine-containing phosphopeptides to study in detail the influence of the modification step on the fragmentation process. MDA modification dramatically improved the fragmentation behavior of peptides that exhibited only one or two dominant cleavages in their unmodified form. Neutral loss of phosphoric acid from phosphopeptides carrying phosphoserine and threonine residues was significantly reduced in favor of a higher abundance of fragment ions. Complementary experiments were carried out on three different instrumental platforms (triple-quadrupole, 3D ion trap, quadrupole-linear ion trap hybrid) to ascertain that the observation is a general effect.     

Fragmentation chemistry of [M + Cu]+ peptide ions containing an N-terminal arginine

[M + Cu]+ peptide ions formed by matrix-assisted laser desorption/ionization from direct desorption off a copper sample stage have sufficient internal energy to undergo metastable ion dissociation in a time-of-flight mass spectrometer. On the basis of fragmentation chemistry of peptides containing an N-terminal arginine, we propose the primary Cu+ ion binding site is the N-terminal arginine with Cu+ binding to the guanidine group of arginine and the N-terminal amine. The principal decay products of [M + Cu]+ peptide ions containing an N-terminal arginine are [a(n) + Cu - H]+ and [b(n) + Cu - H]+ fragments. We show evidence to suggest that [a(n) + Cu - H]+ fragment ions are formed by elimination of CO from [b(n) + Cu - H]+ ions and by direct backbone cleavage. We conclude that Cu+ ionizes the peptide by attaching to the N-terminal arginine residue; however, fragmentation occurs remote from the Cu+ ion attachment site involving metal ion promoted deprotonation to generate a new site of protonation. That is, the fragmentation reactions of [M + Cu]+ ions can be described in terms of a "mobile proton" model. Furthermore, proline residues that are adjacent to the N-terminal arginine do not inhibit formation of [b(n) + Cu - H]+ ion, whereas proline residues that are distant to the charge carrying arginine inhibit formation of [b(n) + Cu - H]+ ions. An unusual fragment ion, [c(n) + Cu + H]+, is also observed for peptides containing lysine, glutamine, or asparagine in close proximity to the Cu+ carrying N-terminal arginine. Mechanisms for formation of this fragment ion are also proposed.     

Electron transfer dissociation with supplemental activation to differentiate aspartic and isoaspartic residues in doubly charged peptide cations

Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide ions allows differentiation of isoaspartic acid and aspartic acid residues using the c + 57 or z ^• − 57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately, the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak, [M-60], for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues.     

Effects of cysteic acid groups on the gas-phase reactivity and dissociation of [M + 4H]4+ ions from insulin chain B

Gas-phase ion/molecule reactions and collision-induced dissociation (CID) were conducted on [M + 4H]4+ of insulin chain B. This Fourier transform mass spectrometry work involved ions from the oxidized peptide (with two cysteic acid residues) and its reduced form (with two cysteine residues). Kinetic behavior during deprotonation and hydrogen/deuterium exchange reactions indicates that insulin B (ox) ions have two distinct structural types. In contrast, insulin B (red) ions have only one major reacting population, which has a more compact structure than the oxidized ions. No significant differences in fragmentation patterns for the two insulin B (ox) populations were observed when CID was performed as a function of deprotonating reaction time. However, markedly different fragmentation was found between [M + 4H]4+ of insulin B (ox) and (red). Therefore, the presence of cysteic acid groups in insulin B (ox) significantly impacts dissociation and presumably structure. This suggests that some insulin B (ox) ions are zwitterionic, with the five basic sites protonated and one cysteic acid group deprotonated. Molecular dynamics calculations revealed several viable structures that are consistent with the experimental results. For example, the most stable form of the reduced ion, which is unprotonated at the His10, is very compact and has lost the alpha-helix of native insulin. Low energy structures for the oxidized ions include a zwitterion with an intraionic interaction between anionic Cyx7 and cationic His10, as well as a nonzwitterionic conformer that lacks a proton at Phe1; both structures retain the alpha-helix. These structures may account for the two experimentally observed isomers, although others are possible. In addition, experiments on oxidized insulin B were conducted from methanolic solution, which may denature the conformation, and pure aqueous solution, which may leave a native conformation. These differences in solvent composition had no effect on the gas-phase results.     

Decompositions of cationized heterodimers of amino acids in relation to charge location in peptide ions

The unimolecular decompositions of protonated heterodimers of native and derivatized amino acids to yield the protonated monomers were studied as a guide to charge location in peptide ions. Analyses using a hybrid instrument of BEqQ geometry demonstrated the advantages (with respect to mass resolution, sensitivityr reproducibility, and the elimination of extraneous signals) of the detection of product ions formed in the radiofrequency-only quadrupole region (q) rather than in the field-free region between Band E. Conversion of arginine to dimethylpyrimidylomithine (DMPO) reduced the proton affinity, as evidenced by the decomposition of the protonated arginine/DMPO heterodimer. Conversion of cysteine to pyridylethylcysteine enhanced the proton affinity. Application of these derivatization procedures to peptides resulted in changes in the observed fragmentations of the protonated precursors consistent with the predicted modifications in charge location. Unimolecular decomposition of the protonated dimer composed of glycine and N-acetylglycine yielded both protonated monomers with abundances differing by a factor of only 2; this suggests that in protonated peptides, the amide bonds are competitive with the N-terminal amino group as sites of protonation. It is clear that the propensities to proton’ or metal-cation location at particular sites in peptides are influenced by both short- and long-range intraionic interactions. In peptides composed of amino acids of similar cation affinities, it may be postulated that the ion population is heterogeneous with respect to the site of charge, with consequent promotion of multiple low-energy fragmentation routes.     

Differentiating alpha- and beta-aspartic acids by electrospray ionization and low-energy tandem mass spectrometry

Spectra obtained by low-energy electrospray ionization tandem mass spectrometry (ESI-MS/MS) of 34 peptides containing aspartic acids at position n were studied and unambiguously differentiated. beta-Aspartic acid yields an internal rearrangement similar to that of the C-terminal rearrangements of protonated and cationized peptides. As a result of this rearrangement, two different ions containing the N- and the C-terminal ends of the original peptide are formed, namely, the bn-1 + H2O and y"l - n + 1 - 46 ions, respectively, where e is the number of amino acid residues in the peptide. The structure suggested for the y"l - n + 1 - 46 ion is identical to that proposed for the vn ions observed upon high-energy collision-induced dissociation (CID) experiments. The intensity of these ions in the low-energy MS/MS spectra is greatly influenced by the presence and position of basic amino acids within the sequences. Peptides with a basic amino acid residue at position n - 1 with respect to the beta-aspartic acid yield very intense bn-1 + H2O ions, while the y"l - n + 1 - 46 ion was observed mostly in tryptic peptides. Comparison between the high- and low-energy MS/MS spectra of several isopeptides suggests that a metastable fragmentation process is the main contributor to this rearrangement, whereas for long peptides (40 AA) CID plays a more important role. We also found that alpha-aspartic acid containing peptides yield the normal immonium ion at 88 Da, while peptides containing beta-aspartic acid yield an ion at m/z 70, and a mechanism to explain this phenomenon is proposed. Derivatizing isopeptides to form quaternary amines, and performing MS/MS on the sodium adducts of isopeptides, both improve the relative intensity of the bn + 1 + H2O ions. Based on the above findings, it was possible to determine the isomerization sites of two aged recombinant growth proteins.     

Advantages of derivatization by osmium tetroxide and 2,2'-bipyridine for matrix-assisted laser desorption/ionization post-source decay fragment ion analysis of peptides

Matrix-assisted laser desorption/ionization--post-source decay (MALDI-PSD) fragment ion analysis is frequently used for peptide sequence determination. PSD fragmentation is often changed or improved in terms of, e.g., sequence coverage, after derivatization. In this work, the influence of modification by an osmium tetroxide-bipyridine reagent (Os,bipy) on the MALDI-PSD behaviour of peptides is studied. The reagent modifies peptides specifically at tryptophan residues and oxidizes methionine to methionine sulfone and cysteine to cysteic acid. As a result the masses of some of the fragments are specifically shifted in case of peptides containing a methionine by +32 Da and, in cases of peptides containing a cysteine residue, by +48 Da. In addition, due to the change in protonation properties of a peptide after oxidation, fragments containing cysteic acid are in most cases totally suppressed. This effect significantly facilitates peptide sequence determination. Improvement of MALDI-TOFMS and PSD analysis after the reaction with Os,bipy is demonstrated for examples involving derivatives of humanin, a novel neuroprotective peptide.     

Origin and Prediction of Highly Specific Bond Cleavage Sites in the Thermal Activation of Intact Protein Ions

Predicting the fragmentation patterns of proteins would be beneficial for the reliable identification of intact proteins by mass spectrometry. However, the ability to accurately make such predictions remains elusive. An approach to predict the specific cleavage sites in whole proteins resulting from collision-induced dissociation by use of an improved electrostatic model for calculating the proton configurations of highly-charged protein ions is reported. Using ubiquitin, cytochrome c, lysozyme and β-lactoglobulin as prototypical proteins, this approach can be used to predict the fragmentation patterns of intact proteins. For sufficiently highly charged proteins, specific cleavages occur near the first low-basicity amino acid residues that are protonated with increasing charge state. Hybrid QM/QM′ (QM=quantum mechanics) and molecular dynamics (MD) simulations and energy-resolved collision-induced dissociation measurements indicated that the barrier to the specific dissociation of the protonated amide backbone bond is significantly lower than competitive charge remote fragmentation. Unlike highly charged peptides, the protons at low-basicity sites in highly charged protein ions can be confined to a limited sequence of low-basicity amino acid residues by electrostatic repulsion, which results in highly specific fragmentation near the site of protonation. This research suggests that the optimal charge states to form specific sequence ions of intact proteins in higher abundances than the use of less specific ion dissociation methods can be predicted a priori.     

Proton transfer reactions for improved peptide characterisation

The combination of deprotonation (via ion/molecule and ion/ion reactions) and low-energy collision-induced dissociation (CID) has been explored for the enhanced characterisation of tryptic peptides via access to different precursor charge states. This approach allows instant access to fragmentation properties of singly and doubly protonated precursors (arising from the availability of mobile protons) in a single experiment. Considering both charge states extended our base of structurally informative data (in comparison with considering just a single charge state) due to generation of additional sequence ions and by obtaining supplementary structural information derived from selective cleavages. Roughly 37% of combined data sets (CID spectra of doubly and singly charged precursor) showed a greater database identification confidence than each set alone. Moreover, comparison between a number of sequence ions of the singly charged precursor and the doubly charged precursor provided a mean of distinguishing the two classes of tryptic peptides (arginine or lysine containing).     

Formation of c1 fragment ions in collision-induced dissociation of glutamine-containing peptide ions: a tip for de novo sequencing

A c1 ion was observed with significant yield in the tandem mass (MS/MS) spectra of peptide ions containing glutamine as the second amino acid residue from the N-terminus. The c1 fragment was generated independently of the N-terminal residue of the peptide, but its abundance was strongly dependent on the side-chain identity. This ion is not a common fragmentation product in low-energy collision-induced dissociation of peptide ions, but it assists in identification of the first two amino acid residues, often difficult due to a low or absent signal from the heaviest y ion. A consecutive fragmentation mechanism is proposed, involving a b2 ion with a six-membered ring as an intermediate, to explain the exceptional stability of the c1 fragment ion. The utility of this information is discussed, especially in de novo sequencing of peptide ions.     

Location of alkali metal binding sites in endothelin A selective receptor antagonists, cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) and cyclo(D-Trp-D-Asp-Pro-D-Ile-Leu), from multistep collisionally activated decompositions

We previously showed by using mass spectrometry that endothelin A selective receptor antagonists BQ123 and JKC301 form novel coordination compounds with sodium ions. This property may underlie the ability of an ET(A) antagonist to induce net tubular sodium reabsorption in the proximal tubule cells and reverse acute renal failure induced by severe ischemia. We have now defined the metal binding sites on BQ123 and JKC301 by subjecting the metal-containing peptides to multiple stages of collisionally activated decomposition (CAD) in an ion trap mass spectrometer. When submitted to low-energy CAD, the ring opens at the Asp-Pro amide bond. The metal ion, which bonds, inter alia, to the carbonyl oxygen of the proline residue, acts as a fixed charge site, and directs a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues are sequentially cleaved from the C-terminal end, and the terminal aziridinone structure moves one step toward the N-terminus with each C-terminal amino acid residue removed. These observations are the basis of a new method to sequence cyclic peptides. Amino acid residues are observed as sets of three ions, a*(n)PD, b*(n)PD and c*(n)PD where n is the number of amino acid residues in the peptide.     

Infrared spectroscopy of cationized arginine in the gas phase: direct evidence for the transition from nonzwitterionic to zwitterionic structure

The gas-phase structures of protonated and alkali metal cationized arginine (Arg) and arginine methyl ester (ArgOMe) are investigated with infrared spectroscopy and ab initio calculations. Infrared spectra, measured in the hydrogen-stretch region, provide compelling evidence that arginine changes from its nonzwitterionic to zwitterionic form with increasing metal ion size, with the transition in structure occurring between lithium and sodium. For sodiated arginine, evidence for both forms is obtained from spectral deconvolution, although the zwitterionic form is predominant. Comparisons of the photodissociation spectra with spectra calculated for low-energy candidate structures provide additional insights into the detailed structures of these ions. Arg*Li+, ArgOMe*Li+, and ArgOMe*Na+ exist in nonzwitterionic forms in which the metal ion is tricoordinated with the amino acid, whereas Arg*Na+ and Arg*K+ predominately exist in a zwitterionic form where the protonated side chain donates one hydrogen bond to the N terminus of the amino acid and the metal ion is bicoordinated with the carboxylate group. Arg*H+ and ArgOMe*H+ have protonated side chains that form the same interaction with the N terminus as zwitterionic, alkali metal cationized arginine, yet both are unambiguously determined to be nonzwitterionic. Calculations indicate that for clusters with protonated side chains, structures with two strong hydrogen bonds are lowest in energy, in disagreement with these experimental results. This study provides new detailed structural assignments and interpretations of previously observed fragmentation patterns for these ions.     

On the maximum charge state and proton transfer reactivity of peptide and protein ions formed by electrospray ionization

A relatively simple model for calculation of the energetics of gas-phase proton transfer reactions and the maximum charge state of multiply protonated ions formed by electrospray ionization is presented. This model is based on estimates of the intrinsic proton transfer reactivity of sites of protonation and point charge Coulomb interactions. From this model, apparent gas-phase basicities (GB^app) of multiply protonated ions are calculated. Comparison of this value to the gas-phase basicity of the solvent from which an ion is formed enables a maximum charge state to be calculated. For 13 commonly electrosprayed proteins, our calculated maximum charge states are within an average of 6% of the experimental values reported in the literature. This indicates that the maximum charge state for proteins is determined by their gas-phase reactivity. Similar results are observed for peptides with many basic residues. For peptides with few basic residues, we find that the maximum charge state is better correlated to the charge state in solution. For low charge state ions, we find that the most basic sites Arg, Lys, and His are preferentially protonated. A significant fraction of the less basic residues Pro, Trp, and Gln are protonated in high charge state ions. The calculated GB^app of individual protonation sites varies dramatically in the high charge state ions. From these values, we calculate a reduced cross section for proton transfer reactivity that is significantly lower than the Langevin collision frequency when the GB^app of the ion is approximately equal to the GB of the neutral base.     

Gas-phase fragmentation characteristics of benzyl-aminated lysyl-containing tryptic peptides

The fragmentation characteristics of peptides derivatized at the side-chain ε-amino group of lysyl residues via reductive amination with benzaldehyde have been examined using collision-induced dissociation (CID) tandem mass spectrometry. The resulting MS/MS spectra exhibit peaks representing product ions formed from two independent fragmentation pathways. One pathway results in backbone fragmentation and commonly observed sequence ion peaks. The other pathway corresponds to the unsymmetrical, heterolytic cleavage of the C_ζ-N_ε bond that links the benzyl derivative to the side-chain lysyl residue. This results in the elimination of the derivative as a benzylic or tropylium carbocation and a (n − l)⁺-charged peptide product (where n is the precursor ion charge state). The frequency of occurrence of the elimination pathway increases with increasing charge of the precursor ion. For the benzylmodified tryptic peptides analyzed in this study, peaks representing products from both of these pathways are observed in the MS/MS spectra of doubly-charged precursor ions, but the carbocation elimination pathway occurs almost exclusively for triply-charged precursor ions. The experimental evidence presented herein, combined with molecular orbital calculations, suggests that the elimination pathway is a charge-directed reaction contingent upon protonation of the secondary ε-amino group of the benzyl-derivatized lysyl side chain. If the secondary ε-amine is protonated, the elimination of the carbocation is observed. If the precursor is not protonated at the secondary ε-amine, backbone fragmentation persists. The application of appropriately substituted benzyl analogs may allow for selective control over the relative abundance of product ions generated from the two pathways.     

A comparison of positive and negative ion collision‐induced dissociation for model heptapeptides with one basic residue

The effects of the identity and position of basic residues on peptide dissociation were explored in the positive and negative modes. Low‐energy collision‐induced dissociation (CID) was performed on singly protonated and deprotonated heptapeptides of the type: XAAAAAA, AAAXAAA, AAAAAXA and AAAAAAX, where X is arginine (R), lysine (K) or histidine (H) residues and A is alanine. For [M + H]⁺, the CID spectra are dominated by cleavages adjacent to the basic residues and the majority of the product ions contain the basic residues. The order of a basic residue's influence on fragmentation of [M + H]⁺ is arginine > histidine ≈ lysine, which is also the order of decreasing gas‐phase basicity for these amino acids. These results are consistent with the side chains of basic residues being positive ion charge sites and with the more basic arginine residues having a higher retention (i.e. sequestering) of the positive charge. In contrast, for [M ? H]^? the identity and position of basic residues has almost no effect on backbone fragmentation. This is consistent with basic residues not being negative mode charge sites. For these peptides, more complete series of backbone fragments, which are important in the sequencing of unknowns, can be found in the negative mode. Spectra at both polarities contain C‐terminal y‐ions, but y_n″⁺ has two more hydrogens than the corresponding y_n^?. Another major difference is the production of the N‐terminal backbone series b_n⁺ in the positive mode and c_n^? in the negative mode. Thus, comparison of positive and negative ion spectra with an emphasis on searching for pairs of ions that differ by 2 Da (y_n″⁺ vs y_n^?) and by 15 Da (b_n⁺ vs c_n^?) may be a useful method for determining whether a product ion is generated from the C‐terminal or the N‐terminal end of a peptide. In addition, a characteristic elimination of NH?C?NH from arginine residues is observed for deprotonated peptides. Copyright © 2010 John Wiley & Sons, Ltd.