Mass spectrometric evidence for mechanisms of fragmentation of charge-derivatized peptides

Mass spectrometry of charged derivatives of peptides has been a growing area of interest in the past decade. Fragmentation of charged derivatives of peptides is believed to be different from than that of protonated peptides when analyzed by collisionally activated dissociation-tandem mass spectrometry (CAD-MS/MS). The charged derivatives fragment by charge-remote fragmentation mechanisms, which are usually classified as high-energy (HE)-CAD processes. Our objective in the present study is to investigate the mechanism of fragmentation of charged derivatives of peptides when analyzed by matrix-assisted laser desorption/ionization-postsource decay-mass spectrometry (MALDI-PSD-MS) and electrospray ionization (ESI)-CAD-MS/MS (ion trap), which involve low-energy processes. Three major types of hydrogens (alpha, beta, and amide) are available for migration during the formation of the *a(n) ions (the predominant ion series produced from these charged derivatives). To pinpoint which of the three hydrogens is involved in the formation of the *a(n) ions, deuterium-labeled peptide derivatives with labels at specific sites were synthesized and analyzed by MALDI-PSD-MS and ESI-CAD-MS/MS. Our results suggest that the amide hydrogen of the residue at which the cleavage occurs shifts during the formation of *a(n); this observation serves as evidence for the mechanism proposed earlier by Liao et al. for fragmentation of such charged derivatives. The results also help elucidate the structure of the *a(n) ions, *b(n) ions, and others formed during cleavage at the proline residue, as well as the ions formed during loss of the C-terminal residue from these charged derivatives.     

Role of the site of protonation in the low-energy decompositions of gas-phase peptide ions

The dissociation of singly or multiply protonated peptide ions by using low-energy collisional activation (CA) is highly dependent on the sites of protonation. The presence of strongly basic amino acid residues in the peptide primary structure dictates the sites of protonation, which generates a precursor ion population that is largely homogeneous with respect to charge sites. Attempts to dissociate this type of precursor ion population by low-energy CA result in poor fragmentation via few pathways. The work described here represents a systematic investigation of the effects of charge heterogeneity in the precursor ion population of a series of model peptides in low-energy CA experiments. Incorporation of acidic residues in the peptide RLC*IFSC*FR (where C* indicates a cysteic acid residue), for example, balances the charge on the basic arginine residues, which enables the ionizing protons to reside on a number of less basic sites along the peptide backbone. This results in a precursor ion population that is heterogeneous with respect to charge site. Low-energy CA of these ions results in diverse and efficient fragmentation. Molecular modeling has been utilized to demonstrate that energetically preferred conformations incorporate an intraionic interaction between arginine and cysteic acid residues.     

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 peptide fragmentation by N-terminal derivatization with high proton affinity

An improved method of de novo peptide sequencing based on mass spectrometry using novel N-terminal derivatization reagents with high proton affinity has been developed. The introduction of a positively charged group into the N-terminal amino group of a peptide is known to enhance the relative intensity of b-ions in product ion spectra, allowing the easy interpretation of the spectra. However, the physicochemical properties of charge derivatization reagents required for efficient fragmentation remain unclear. In this study, we prepared several derivatization reagents with high proton affinity, which are thought to be appropriate for peptide fragmentation under low-energy collision-induced dissociation (CID) conditions, and examined their usefulness in de novo peptide sequencing. Comparison of the effects on fragmentation among three derivatization reagents having a guanidino or an amidino moiety, which differ in proton affinity, clearly indicated that there was an optimal proton affinity for efficient fragmentation of peptides. Among reagents tested in this study, derivatization with 4-amidinobenzoic acid brought about the most effective fragmentation. This derivatization approach will offer a novel de novo peptide sequencing method under low-energy CID conditions.     

Sequencing of sulfonic acid derivatized peptides by electrospray mass spectrometry

We report the application of nanoelectrospray ionization tandem mass spectrometry (nES-MS/MS) and capillary LC/microelectrospray MS/MS (cLC/&mgr;ES-MS/MS) for sequencing sulfonic acid derivatized tryptic peptides. These derivatives were specifically prepared to facilitate low-energy charge-site-initiated fragmentation of C-terminal arginine-containing peptides, and to enhance the selective detection of a single series of y-type fragment ions. Both singly and doubly protonated peptides were analyzed by MS/MS and the results were compared with those from their derivatized counterparts. Model peptides and peptides from tryptic digests of gel-isolated proteins were analyzed. Derivatized singly protonated peptides fragment in the same way by nES-MS/MS as they do by post-source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD-MALDI-MS). They produce fragment ion spectra dominated by y-ions, and the simplified spectra are readily interpreted de novo. Doubly protonated peptides fragment in much the same way as their non-derivatized doubly protonated counterparts. The fragmentation of doubly protonated derivatives is especially useful for sequencing peptides that possess a proline residue near the N-terminus of the molecule. The singly protonated forms of these proline-containing derivatives often show enhanced fragmentation on the N-terminal side of the proline and considerably reduced fragmentation on the C-terminal side. In addition, sulfonic acid derivatization increases the in-source fragmentation of arginine-containing peptides. This could be useful for sequence verification and sequence tagging for use in single stage mass spectrometry. Copyright 2000 John Wiley & Sons, Ltd.     

Five-membered ring formation in unimolecular reactions of peptides: a key structural element controlling low-energy collision-induced dissociation of peptides

Unimolecular fragmentation reactions of peptides in low-energy collision-induced dissociation are reviewed in the mechanistic context of five-membered ring formation. This structure of intermediates or of fragment ions is recognized as a key element that governs unimolecular peptide fragmentation within the structural framework determined by the peptide backbone and its side-chains. A collection of collision-induced dissociation reactions is presented covering (i) b-ion formation, (ii) the fragmentation of N-terminally acylated peptides, (iii) neutral loss of the C-terminal amino acid in alkali or silver cationized peptides, (iv) the fragmentation of isoAsp-containing peptides and (v) the fragmentation of negatively charged Asp- or Glu-containing peptides. It appears that for all possible nucleophile-electrophile interactions leading to a five-membered ring structure an associated unimolecular peptide fragmentation reaction can be observed.     

Combined quantum chemical and RRKM modeling of the main fragmentation pathways of protonated GGG. II. Formation of b(2), y(1), and y(2) ions

Quantum chemical and RRKM calculations were performed on protonated GGG in order to determine the atomic details of the main fragmentation pathways leading to formation of b(2),y(1), and y(2) ions. Formation of y(1) ions on the "diketopiperazine" pathway is initiated from relatively high-energy C-terminal amide nitrogen protonated species for which the N-terminal amide bond is in the cis isomerization state. The reaction goes through a transition structure which is only slightly less favored than the reactive configuration itself. RRKM calculations indicate that this reaction is extremely fast as soon as the fragmenting species have more internal energy than the reaction threshold. The calculated energetics suggests that y(1) ions are formed on the "diketopiperazine" pathway with a non-negligible (6-10 kcal/mol) reverse activation barrier. Investigation of species occurring during the formation of b(2) ions having an oxazolone structure indicates that y(1) ions can be formed also from intermediates previously thought to result in only b(2) ions. As the first step of the "b(x)-y(z)" pathway proposed here the extra proton must reach the nitrogen of the C-terminal amide bond. Attack of the N-terminal amide oxygen on the carbon center of the C-terminal amide bond results in formation of the oxazolone ring while the detaching G leaves the precursor ion. Under low-energy collision conditions the complex of protonated 2-aminomethyl-5-oxazolone and G can rearrange to form a proton-bonded dimer of these species. In such circumstances the extra proton is shared by the two monomers and dissociation of the dimer will be determined by the thermochemistry involved. Based on the "b(x)-y(z)" pathway one can easily explain the linear relationship between the logarithm of the y(1)/b(2) ion abundance ratio and the proton affinity of the C-terminal amino acid substituent for the series of H-Gly-Gly-Xxx-OH tripeptides where Xxx was varied (Morgan DG, Bursey MM. Org. Mass. Spectrom. 1994; 29: 354). The calculated energetics indicates that both y(1) and b(2) ions are formed with no reverse activation barrier on the "b(x)-y(z)" pathway.     

Effect of electrospray ionization conditions on low-energy tandem mass spectra of peptides

The effect of electrospray ionization (ESI) conditions on low-energy tandem mass spectra of peptides in the relative molecular mass range 400–1200 was examined. For singly charged peptide ions the source skimmer potential (which determines the degree of acceleration of the ions through the intermediate pressure region in the source) can strongly influence the extent of fragmentation observed in tandem mass spectra, especially at low collision energies. For each peptide there is an optimum skimmer potential which represents a balance between generating ions with sufficient internal energy for subsequent tandem mass spectrometric experiments and inducing the onset of other processes such as source fragmentation. The fragmentation which can be achieved in tandem mass spectra with high skimmer potentials differs from ESI source fragmentation for the same peptides. We have found that fragmentation in ESI mass spectra depends both on skimmer potential and on solvent pH, presumably because the latter determines the proportion of doubly charged species generated from a given peptide. Low-energy tandem mass spectra of peptides following ESI are equally as sensitive to peptide structure and the type of adduct studied (e.g. [M + H]⁺ vs. [M + NH₄]⁺) as tandem mass spectra obtained following older ionization methods such as fast atom bombardment.     

Leaving group effects on the selectivity of the gas-phase fragmentation reactions of side chain fixed-charge-containing peptide ions

The effect of trialkylsulfonium versus quaternaryalkylammonium ions on the multistage gas-phase fragmentation reactions of side chain, fixed-charge, cysteine-containing peptides has been examined in a quadrupole linear ion trap. These tandem mass spectrometry experiments reveal that selective loss of dialkylsulfide from fixed-charge sulfonium ion derivatives is the dominant fragmentation pathway regardless of the degree of proton mobility, indicating that it is the most energetically favored fragmentation pathway. In contrast, the loss of trimethylamine from the side chain of fixed-charge ammonium-ion-containing cysteine derivatives appears to be less energetically favored, and as a result extensive charge-remote fragmentation is observed under low proton mobility conditions, while under conditions of high proton mobility, amide bond fragmentation reactions dominate. These findings are supported by molecular orbital calculations at the B3LYP/6-31 + G(d, p) level of theory, which showed that the neutral loss of dimethylsulfide is both thermodynamically and kinetically preferred over the loss of trimethylamine.     

Formation of [b(n−1)+OH+H]+ ion structural analogs by solution-phase chemistry

Derivatization of a variety of peptides by a method known to enhance anhydride formation is demonstrated by mass spectrometry to yield ions that have elemental composition and fragmentation properties identical to [b(n-1) + OH + H]+ ions formed by gas-phase rearrangement and fragmentation. The [b(n-1) + OH + H]+ ions formed by gas-phase rearrangement and fragmentation and the solution-phase [b(n-1) + OH + H]+ ion structural analogs formed by derivatization chemistry show two different forms of dissociation using multiple-collision CAD in a quadrupole ion trap and unimolecular decomposition in a TOF-TOF; one group yields identical product ions as a truncated form of the peptide with a free C-terminal carboxylic acid and fragments at the same activation energy; the other group fragments differently from the truncated peptide, being more resistant to fragmentation than the truncated peptide and yielding primarily the [b(n-2) + OH + H]+ product ion. Nonergodic electron capture dissociation MS/MS suggests that any structural differences between the specific-fragmenting [b(n-1) + OH + H]+ ions and the truncated peptide is at the C-terminus of the peptide. The specific-fragmentation can be readily observed by MS(n) experiments to occur in an iterative fashion, suggesting that the C-terminal structure of the original [b(n-1) + OH + H]+ ion is maintained after subsequent rearrangement and fragmentation events in peptides which fragment specifically. A mechanism for the formation of specific-fragmenting and nonspecific-fragmenting [b(n-1) + OH + H]+ ions is proposed.     

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.     

The effect of charge state and the localization of charge on the collision-induced dissociation of peptide ions

The effect that charge state has on the collision-induced dissociation (CID) of peptide ions is examined in detail for several representative peptides under high-energy collision conditions. The CID spectra of singly and doubly charged precursor ions (generated by fast-atom bombardment and electrospray ionization, respectively) are compared for several peptides with similar primary structure. It is shown that for peptides that contain highly basic amino acids, the dissociation of doubly charged ions is strongly influenced by the position of these residues within the peptide and the general observations reported concerning the dissociation of singly charged ions can be extended to precursors with higher charge states. Based on the dissociation behavior of the doubly charged ions of these peptides, it is demonstrated that two charges can reside in close proximity in the precursor ions, overcoming possible repulsion effects, when favored by a high concentration of basic sites. In addition)’ this work illustrates that in the case of doubly charged ions..the charge state of some fragment ions can be determined directly from the mass-to-charge ratio assignments of the CID spectrum.     

Metastable decomposition of peptide [M + H]+ ions via rearrangement involving loss of the C-terminal amino acid residue

A novel fragmentation of metastable peptide [M + H]⁺ ions is described. Loss of the C-terminal amino acid residue is accomqanied by retention of one of the carboxyl oxygens, as judged by ¹⁸O-labeling. The retained ⁸O label is located at the new C-terminus. Sequential mass spectrometric analyses indicate that the structure of the first-generation product ion is indistinguishable from that of the [M + H]⁺ ion of the peptide with one fewer amino acid residues. Thus, for example, the metastable decompositions of ions of m/z 904 are similar whether they correspond to des-Arg⁹-bradykinin [M + H]⁺ ions or to fragments derived from bradykinin [M + H]⁺ ions. No corresponding rearrangements have been observed for peptides with C-terminal amide or ester functions. The mechanism of this fragmentation may be considered to be analogous to that previously suggested for fragmentations of [M + alkali metal cation]⁺ ions. For the examples of bradykinin and related peptides, the rearrangement is strongly promoted when arginine is the amino acid residue lost. The same fragmentation is also favored by the presence of an arginine residue at or near the N-terminus. The strong influence of peptide amino acid composition, including residues remote from the C-terminus, on the prevalence of this fragmentation suggests mechanistic complexities that require further elucidation.

     

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

The fragmentation of positive and negative ions of peptide disulfides under mass spectrometric conditions yields distinctly different product ion distributions. A negative ion upon collision induced dissociation yields intense product ions, which correspond to cleavage at the disulfide linkage. The complete assignment of the product ions obtained upon fragmentation of oxidized glutathione in an ion trap is presented. The cleavage at the disulfide site is mediated by abstraction of CalphaH and CbetaH protons resulting in product ions derived by neutral loss of H2S2 and H2S. The formation of peptide thioaldehydes and persulfides at the cysteine sites is established. Dehydroalanine formation at the Cys residue is predominant. The case of a contryphan, a cyclic peptide disulfide derived from Conus snail venom, illustrates the utility of negative ion mass spectrometry in disulfide identification. Complementary information is derived by combining the fragmentation patterns obtained from positive and negative ions of disulfide containing peptides.     

Fragmentation mechanisms of oligodeoxynucleotides: Effects of replacing phosphates with methylphosphonates and thymines with other bases in T-rich sequences

This article reports another step in an ongoing effort to understand the fragmentation of T-rich oligodeoxynucleotides. We extended an earlier investigation of T-rich 4-mers to T-rich 6-mers, 8-mers and 10-mers by using four different tandem mass spectrometric methods. The methods include low-energy collisionally activated decomposition (CAD) of electrospray ionization (ESI)-produced ions, source-CAD of ESI-produced ions, post-source decay (PSD), and CAD of matrix assisted laser desorption ionization (MALDI)-generated ions. The most abundant fragment ions produced from [M - 2H]2- precursors upon low-energy CAD in an ion trap are the [a - B]- and their complementary w ions. The predominant cleavage sites for T-rich oligodeoxynucleotides are always the 3' C-O bonds adjoining a non-T nucleobase (i.e., a base with a higher proton affinity (PA) than that of T). The relative abundance of [a - B]- correlates with the PAs of the nucleobases, underscoring the importance of proton transfer to the base. The propensity to form [a - B]- ions falls in the order of G > C approximately A > T. Structural isomers up to 10-mers can be readily sequenced and distinguished with each of the four tandem mass spectrometric methods applied. The fragmentation of oligodeoxynucleotides in which various phosphates were replaced with methylphosphonate is a measure of the participation of the phosphate proton in the formation of [a - B] ions. For 4 and 5-mers, transfer of an acidic proton from the 5'-phosphate to the departing base is the initiating step in the formation of [a - B]- ions.     

Influences of peptide side chains on the metal ion binding site in metal ion-cationized peptides: Participation of aromatic rings in metal chelation

Aromatic side chains on amino acids influence the fragmentations of cationic complexes of doubly charged metal ions and singly deprotonated peptides. The metal ion interacts with an aromatic side chain and binds to adjacent amide nitrogens. When fragmentation occurs, this bonding leads to the formation of abundant metal-containing a-type ions by reactions that occur at the sites of amino acids that contain the aromatic side chain. Furthermore, formation of metal-containing immonium ions of the amino acids that contain the aromatic side chain also are formed. The abundant a-type ions may be useful in interpretation strategies in which it is necessary to locate in a peptide the position of an amino acid that bears an aromatic side chain.     

Investigation of intramolecular proton migration in a series of model, metal-cationized tripeptides using in situ generation of an isotope label

In this study we used an isotope label, generated in situ, to investigate intramolecular proton migration or scrambling during formation of [b(2)+17+Li](+) products by collision-induced dissociation (CID) of Li(+)-cationized tripeptides. To generate the isotope label, we used a McLafferty-type rearrangement of N-terminally acetylated, C-terminal peptide tert-butyl esters in which all amide positions were exchanged with deuterium. Using a set of small, model peptides, we show that intramolecular proton scrambling occurs during CID, particularly amongst adjacent sites along a peptide backbone, on the time scales employed for low-energy collisional activation in an ion-trap mass spectrometer.     

Charge stripping and the site of cationization of substituted aromatic compounds

The site of protonation, methylation and ethylation of anilines, phenols and thiophenols can be determined by tandem mass spectrometry (MS/MS). The extent to which the cation attachment products formed from these compounds undergo charge stripping is related to the site of cation attachment. The formation of doubly charged ions for phenols and anilines is favored when cation attachment occurs on the ring. For thiophenols alkylation occurs almost exclusively on the substituent, and yet an abundant stripping peak is recorded. This behavior is opposite to that seen for anilines and phenols and is accounted for in terms of orbital energies in the three systems. The site of cation attachment, as deduced by charge stripping, agrees with conclusions drawn from the fragmentation behavior of these ions upon collision-induced dissociation.     

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.     

Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (b_n-1 + H₂O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H₂O and NH₃) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in the high mass range of the MS/MS spectra. The mass difference between this signal and the protonated molecular ion corresponds to the mass of the C-terminal residue. It allowed a straightforward identification of the amino acid positioned at this extremity. It must be emphasized that a neutral residue loss can be misattributed to the formation of a y_m-1 ion, i.e., to the loss of the N-terminal residue following the a₁-y_m–1 fragmentation channel. Extreme caution must be adopted when reading the direct sequence ion on the positive ion MS/MS spectra of singly charged peptides not to mix up the attribution of the N- and C-terminal amino acids. Although such peculiar fragmentation behavior is of obvious interest for de novo peptide sequencing, it can also be exploited in proteomics, especially for studies involving digestion protocols carried out with proteolytic enzymes other than trypsin (Lys-N, Glu-C, and Asp-N) that produce arginine-containing peptides.