The effect of radical trap moieties on electron capture dissociation spectra of substance P

To further test the hypothesis that electron capture dissociation (ECD) involves long-lived radical intermediates and radical migration occurs within these intermediates before fragmentation, radical trap moieties were attached to peptides with the assumption that they would reduce fragmentation by decreasing the mobility of the radical. Coumarin labels were chosen for the radical traps, and unlabeled, singly-labeled, and doubly-labeled Substance P were analyzed by ECD. The results demonstrated a correlation between the number and position of tags on the peptide and the intensity of side-chain cleavages observed, as well as an inverse correlation between the number of tags on the peptide and the intensity of backbone cleavages. Addition of radical traps to the peptide inhibits backbone cleavages, suggesting that either radical mobility is required for these cleavages, or new noncovalent interactions prevent separation of backbone cleavage fragments. The enhancement of side-chain cleavages and the observation of new side-chain cleavages associated with aromatic groups suggest that the gas-phase conformation of this peptide is substantially distorted from untagged Substance P and involves previously unobserved interactions between the coumarin tags and the phenylalanine residues. Furthermore, the use of a double resonance (DR)-ECD experiment showed that these side-chain losses are all products of long-lived radical intermediate species, which suggests that steric hindrance prevents the coumarin-localized radical from interacting with the backbone while simultaneously increasing the radical rearrangements with the side chains.     

The effect of fixed charge modifications on electron capture dissociation

Electron capture dissociation (ECD) studies of two modified amyloid beta peptides (20-29 and 25-35) were performed to investigate the role of H* radicals in the ECD of peptide ions and the free-radical cascade (FRC) mechanism. 2,4,6-Trimethylpyridinium (TMP) was used as the fixed charge tag, which is postulated to both trap the originally formed radical upon electron capture and inhibit the H* generation. It was found that both the number and locations of the fixed charge groups influenced the backbone and side-chain cleavages of these peptides in ECD. In general, the frequency and extent of backbone cleavages decreased and those of side-chain cleavages increased with the addition of fixed charge tags. A singly labeled peptide with the tag group farther away from the protonated site experienced a smaller abundance decrease in backbone cleavage fragments than the one with the tag group closer to the protonated site. Despite the nonprotonated nature of all charge carriers in doubly labeled peptide ions, several c and z* ions were still observed in their ECD spectra. Thus, although H* transfer may be important for the NC(alpha) bond cleavage, there also exist other pathways, which would require a radical migration via H* abstraction through space or via an amide superbase mechanism. Finally, internal fragment ions were observed in the ECD of these linear peptides, indicating that the important role of the FRC in backbone cleavages is not limited to the ECD of cyclic peptides.     

Characterization of amino acid side chain losses in electron capture dissociation

We have used electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry to characterize amino acid side chain losses observed during electron capture dissociation (ECD) of ten 7- to 14-mer peptides. Side-chain cleavages were observed for arginine, histidine, asparagine or glutamine, methionine, and lysine residues. All peptides containing an arginine, histidine, asparagine or glutamine showed the losses associated with that residue. Methionine side-chain loss was observed for doubly-protonated bombesin. Lysine side-chain loss was observed for triply-protonated dynorphin A fragment 1-13 but not for the doubly-protonated ion. The proximity of arginine to a methoxy C-terminal group significantly enhances the extent of side-chain fragmentation. Fragment ions associated with side-chain losses were comparable in abundance to those resulting from backbone cleavage in all cases. In the ECD spectrum of one peptide, the major product was due to fragmentation within an arginine side chain. Our results suggest that cleavages within side chains should be taken into account in analysis of ECD mass spectral data. Losses from arginine, histidine, and asparigine/glutamine can be used to ascertain their presence, as in the analysis of unknown peptides, particularly those with non-linear structures.     

Charge remote fragmentation in electron capture and electron transfer dissociation

Secondary fragmentations of three synthetic peptides (human αA crystallin peptide 1-11, the deamidated form of human βB2 crystallin peptide 4-14, and amyloid β peptide 25-35) were studied in both electron capture dissociation (ECD) and electron-transfer dissociation (ETD) mode. In ECD, in addition to c and z· ion formations, charge remote fragmentations (CRF) of z· ions were abundant, resulting in internal fragment formation or partial/entire side-chain losses from amino acids, sometimes several residues away from the backbone cleavage site, and to some extent multiple side-chain losses. The internal fragments were observed in peptides with basic residues located in the middle of the sequences, which was different from most tryptic peptides with basic residues located at the C-terminus. These secondary cleavages were initiated by hydrogen abstraction at the α-, β-, or γ-position of the amino acid side chain. In comparison, ETD generates fewer CRF fragments than ECD. This secondary cleavage study will facilitate ECD/ETD spectra interpretation, and help de novo sequencing and database searching.     

Electron Capture Dissociation of Hydrogen-Deficient Peptide Radical Cations

Hydrogen-deficient peptide radical cations exhibit fascinating gas phase chemistry, which is governed by radical driven dissociation and, in many cases, by a combination of radical and charge driven fragmentation. Here we examine electron capture dissociation (ECD) of doubly, [M + H]^2+?, and triply, [M + 2H]^3+?, charged hydrogen-deficient species, aiming to investigate the effect of a hydrogen-deficient radical site on the ECD outcome and characterize the dissociation pathways of hydrogen-deficient species in ECD. ECD of [M + H]^2+? and [M + 2H]^3+? precursor ions resulted in efficient electron capture by the hydrogen-deficient species. However, the intensities of c- and z-type product ions were reduced, compared with those observed for the even electron species, indicating suppression of N?CC_?? backbone bond cleavages. We postulate that radical recombination occurs after the initial electron capture event leading to a stable even electron intermediate, which does not trigger N?CC_?? bond dissociations. Although the intensities of c- and z-type product ions were reduced, the number of backbone bond cleavages remained largely unaffected between the ECD spectra of the even electron and hydrogen-deficient species. We hypothesize that a small ion population exist as a biradical, which can trigger N?CC_?? bond cleavages. Alternatively, radical recombination and N?CC_?? bond cleavages can be in competition, with radical recombination being the dominant pathway and N?CC_?? cleavages occurring to a lesser degree. Formation of b- and y-type ions observed for two of the hydrogen-deficient peptides examined is also discussed.     

Electron-capture induced dissociation of doubly charged dipeptides: on the neutral losses and N-Cα bond cleavages

Electron capture by doubly charged peptide cations leads to neutral losses in addition to N-C(α) bond cleavages that give c and z fragments. In this work we discuss the influence of amino acid sequence on hydrogen versus ammonia loss and the propensity for subsequent partial side-chain cleavage after ammonia loss to give w fragment ions. Experiments were done on two series of doubly protonated dipeptides, [XK+2H](2+) and [XR+2H](2+), where X is one of the twenty common amino acid residues, excluding aspartic acid (D), and K and R are lysine and arginine, respectively. While it was previously established that NH(3) is lost exclusively from the N-terminal ammonium group and not from side-chain ammonium groups, we find here that ammonia can be lost from guanidinium radicals as well. The ratio between H loss and NH(3) loss reveals some information on internal ionic hydrogen bonds and peptide conformation since proton sharing between the N-terminal ammonium group and a basic side chain decreases the probability for NH(3) loss due to a lower recombination energy and as a result reduced capture probability. The abundance of w ions was found to correlate with the reaction energy for their formation; highest yield was found for CK and lowest for AK and HK. The survival rate of charge-reduced species was higher for XR than for XK, which is likely linked to the formation of long-lived C(α) radicals in the latter case. The probability for N-C(α) bond cleavage is smaller on average for XR than for XK which indicates that hydrogen transfer from the ε-ammonium radical to the amide group triggers some of the cleavages, or is a result of the different distances between the amide group and the charges in XR and XK. Finally, our data support the previous concept that charge partitioning between c and z fragments can be explained by competition between the two fragments for the proton.     

Cascade Dissociations of Peptide Cation-Radicals. Part 1. Scope and Effects of Amino Acid Residues in Penta-, Nona-, and Decapeptides

Amino acid residue-specific backbone and side-chain dissociations of peptide z ions in MS(3) spectra were elucidated for over 40 pentapeptides with arginine C-terminated sequences of the AAXAR and AAHXR type, nonapeptides of the AAHAAXX"AR and AAHAXAX"AR type, and AAHAAXX"AAR decapeptides. Peptide z(n) ions containing amino acid residues with readily transferrable benzylic or tertiary β-hydrogen atoms (Phe, Tyr, His, Trp, Val) underwent facile backbone cleavages to form dominant z(n-2) or z(n-3) ions. These backbone cleavages are thought to be triggered by a side-chain β-hydrogen atom transfer to the z ion C(α) radical site followed by homolytic dissociation of the adjacent C(α)-CO bond, forming x(n-2) cation-radicals that spontaneously dissociate by loss of HNCO. Amino acid residues that do not have readily transferrable β-hydrogen atoms (Gly, Ala) do not undergo the z(n) → z(n-2) dissociations. The backbone cleavages compete with side-chain dissociations in z ions containing Asp and Asn residues. Side-chain dissociations are thought to be triggered by α-hydrogen atom transfers that activate the C(β)-C(γ) or C(β)-heteroatom bonds for dissociations that dominate the MS(3) spectra of z ions from peptides containing Leu, Cys, Lys, Met, Ser, Arg, Glu, and Gln residues. The Lys, Arg, Gln, and Glu residues also participate in γ-hydrogen atom transfers that trigger other side-chain dissociations.     

Mobile and localized protons: a framework for understanding peptide dissociation

Protein identification and peptide sequencing by tandem mass spectrometry requires knowledge of how peptides fragment in the gas phase, specifically which bonds are broken and where the charge(s) resides in the products. For many peptides, cleavage at the amide bonds dominate, producing a series of ions that are designated b and y. For other peptides, enhanced cleavage occurs at just one or two amino acid residues. Surface-induced dissociation, along with gas-phase collision-induced dissociation performed under a variety of conditions, has been used to refine the general 'mobile proton' model and to determine how and why enhanced cleavages occur at aspartic acid residues and protonated histidine residues. Enhanced cleavage at acidic residues occurs when the charge is unavailable to the peptide backbone or the acidic side-chain. The acidic H of the side-chain then serves to initiate cleavage at the amide bond immediately C-terminal to Asp (or Glu), producing an anhydride. In contrast, enhanced cleavage occurs at His when the His side-chain is protonated, turning His into a weak acid that can initiate backbone cleavage by transferring a proton to the backbone. This allows the nucleophilic nitrogen of the His side-chain to attack and form a cyclic structure that is different from the 'typical' backbone cleavage structures.     

Characterization of Tyrosine Nitration and Cysteine Nitrosylation Modifications by Metastable Atom-Activation Dissociation Mass Spectrometry

The fragmentation behavior of nitrated and S-nitrosylated peptides were studied using collision induced dissociation (CID) and metastable atom-activated dissociation mass spectrometry (MAD-MS). Various charge states, such as 1+, 2+, 3+, 2–, of modified and unmodified peptides were exposed to a beam of high kinetic energy helium (He) metastable atoms resulting in extensive backbone fragmentation with significant retention of the post-translation modifications (PTMs). Whereas the high electron affinity of the nitrotyrosine moiety quenches radical chemistry and fragmentation in electron capture dissociation (ECD) and electron transfer dissociation (ETD), MAD does produce numerous backbone cleavages in the vicinity of the modification. Fragment ions of nitrosylated cysteine modifications typically exhibit more abundant neutral losses than nitrated tyrosine modifications because of the extremely labile nature of the nitrosylated cysteine residues. However, compared with CID, MAD produced between 66% and 86% more fragment ions, which preserved the labile –NO modification. MAD was also able to differentiate I/L residues in the modified peptides. MAD is able to induce radical ion chemistry even in the presence of strong radical traps and therefore offers unique advantages to ECD, ETD, and CID for determination of PTMs such as nitrated and S-nitrosylated peptides.     

The dehydroalanine effect in the fragmentation of ions derived from polypeptides

The fragmentation of peptides and proteins upon collision‐induced dissociation (CID) is highly dependent on sequence and ion type (e.g. protonated, deprotonated, sodiated, odd electron, etc.). Some amino acids, for example aspartic acid and proline, have been found to enhance certain cleavages along the backbone. Here, we show that peptides and proteins containing dehydroalanine, a non‐proteinogenic amino acid with an unsaturated side‐chain, undergo enhanced cleavage of the N—Cα bond of the dehydroalanine residue to generate c‐ and z‐ions. Because these fragment ion types are not commonly observed upon activation of positively charged even‐electron species, they can be used to identify dehydroalanine residues and localize them within the peptide or protein chain. While dehydroalanine can be generated in solution, it can also be generated in the gas phase upon CID of various species. Oxidized S‐alkyl cysteine residues generate dehydroalanine upon activation via highly efficient loss of the alkyl sulfenic acid. Asymmetric cleavage of disulfide bonds upon collisional activation of systems with limited proton mobility also generates dehydroalanine. Furthermore, we show that gas‐phase ion/ion reactions can be used to facilitate the generation of dehydroalanine residues via, for example, oxidation of S‐alkyl cysteine residues and conversion of multiply‐protonated peptides to radical cations. In the latter case, loss of radical side‐chains to generate dehydroalanine from some amino acids gives rise to the possibility for residue‐specific backbone cleavage of polypeptide ions. Copyright © 2016 John Wiley & Sons, Ltd.     

Electron capture in charge-tagged peptides. Evidence for the role of excited electronic states

Electron capture dissociation (ECD) was studied with doubly charged dipeptide ions that were tagged with fixed-charge tris-(2,4,6-trimethoxyphenyl)phosphonium-methylenecarboxamido (TMPP-ac) groups. Dipeptides GK, KG, AK, KA, and GR were each selectively tagged with one TMPP-ac group at the N-terminal amino group while the other charge was introduced by protonation at the lysine or arginine side-chain groups to give (TMPP-ac-peptide + H)(2+) ions by electrospray ionization. Doubly tagged peptide derivatives were also prepared from GK, KG, AK, and KA in which the fixed-charge TMPP-ac groups were attached to the N-terminal and lysine side-chain amino groups to give (TMPP-ac-peptide-ac-TMPP)(2+) dications by electrospray. ECD of (TMPP-ac-peptide + H)(2+) resulted in 72% to 84% conversion to singly charged dissociation products while no intact charge-reduced (TMPP-ac-dipeptide + H)(+) ions were detected. The dissociations involved loss of H, formation of (TMPP + H)(+), and N-C(alpha) bond cleavages giving TMPP-CH(2)CONH(2)(+) (c(0)) and c(1) fragments. In contrast, ECD of (TMPP-ac-peptide-ac-TMPP)(2+) resulted in 31% to 40% conversion to dissociation products due to loss of neutral TMPP molecules and 2,4,6-trimethoxyphenyl radicals. No peptide backbone cleavages were observed for the doubly tagged peptide ions. Ab initio and density functional theory calculations for (Ph(3)P-ac-GK + H)(2+) and (H(3)P-ac-GK + H)(2+) analogs indicated that the doubly charged ions contained the lysine side-chain NH(3)(+) group internally solvated by the COOH group. The distance between the charge-carrying phosphonium and ammonium atoms was calculated to be 13.1-13.2 A in the most stable dication conformers. The intrinsic recombination energies of the TMPP(+)-ac and (GK + H)(+) moieties, 2.7 and 3.15 eV, respectively, indicated that upon electron capture the ground electronic states of the (TMPP-ac-peptide + H)(+*) ions retained the charge in the TMPP group. Ground electronic state (TMPP-ac-GK + H)(+*) ions were calculated to spontaneously isomerize by lysine H-atom transfer to the COOH group to form dihydroxycarbinyl radical intermediates with the retention of the charged TMPP group. These can trigger cleavages of the adjacent N-C(alpha) bonds to give rise to the c(1) fragment ions. However, the calculated transition-state energies for GK and GGK models suggested that the ground-state potential energy surface was not favorable for the formation of the abundant c(0) fragment ions. This pointed to the involvement of excited electronic states according to the Utah-Washington mechanism of ECD.     

The Radical Ion Chemistry of S-Nitrosylated Peptides

The radical ion chemistry of a suite of S-nitrosopeptides has been investigated. Doubly and triply-protonated ions of peptides NYCGLPGEYWLGNDK, NYCGLPGEYWLGNDR, NYCGLPGERWLGNDR, NACGAPGEKWAGNDK, NYCGLPGEKYLGNDK, NYGLPGCEKWYGNDK and NYGLPGEKWYGCNDK were subjected to electron capture dissociation (ECD), and collision-induced dissociation (CID). The peptide sequences were selected such that the effect of the site of S-nitrosylation, the nature and position of the basic amino acid residues, and the nature of the other amino acid side chains, could be interrogated. The ECD mass spectra were dominated by a peak corresponding to loss of ^?NO from the charge-reduced precursor, which can be explained by a modified Utah-Washington mechanism. Some backbone fragmentation in which the nitrosyl modification was preserved was also observed in the ECD of some peptides. Molecular dynamics simulations of peptide ion structure suggest that the ECD behavior was dependent on the surface accessibility of the protonated residue. CID of the S-nitrosylated peptides resulted in homolysis of the S?CN bond to form a long-lived radical with loss of ^?NO. The radical peptide ions were isolated and subjected to ECD and CID. ECD of the radical peptide ions provided an interesting comparison to ECD of the unmodified peptides. The dominant process was electron capture without further dissociation (ECnoD). CID of the radical peptide ions resulted in cysteine, leucine, and asparagine side chain losses, and radical-induced backbone fragmentation at tryptophan, tyrosine, and asparagine residues, in addition to charge-directed backbone fragmentation.     

Electron-Capture and -Transfer Dissociation of Peptides Tagged with Tunable Fixed-Charge Groups: Structures and Dissociation Energetics

Pyridiniummethylcarbonyl moieties that were previously designed on the basis of electronic structure analysis are now utilized as fixed-charge tags with tunable electronic properties to be used for N-terminal peptide derivatization and sequencing by electron-transfer dissociation. Dipeptides AK and KA were derivatized at the peptide N-terminus with 4-dimethylaminopyridinium-N-acetyl (DMAP-ac) and pyridinium-N-acetyl (pyrid-ac) tags of increasing intrinsic recombination energies. Upon the capture of a free electron or electron transfer from fluoranthene anions, (DMAP-ac-AK+H)²⁺, (DMAP-ac-KA+H)²⁺, (pyrid-ac-AK+H)²⁺ and (pyrid-ac-KA+H)²⁺ ions, as well as underivatized (AK+2H)²⁺, completely dissociated. The fixed-charge tags steered the dissociation upon electron transfer to form abundant backbone N–C_α bond cleavages, whereas the underivatized peptide mainly underwent H-atom and side-chain losses. Precursor ion structures for the tagged peptides were analyzed by an exhaustive conformational search combined with B3LYP/6-31+G(d,p) geometry optimization and single-point energy calculations in order to select the global energy minima. Structures, relative energies, transition states, ion–molecule complexes, and dissociation products were identified for several charge-reduced species from the tagged peptides. The electronic properties of the charge tags and their interactions with the peptide moieties are discussed. Electrospray ionization and electron-transfer dissociation of larger peptides are illustrated with a DMAP-tagged pentapeptide.     

Unusual Fragmentation of β-Linked Peptides by ExD Tandem Mass Spectrometry

Ion-electron reaction based fragmentation methods (ExD) in tandem mass spectrometry (MS), such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) represent a powerful tool for biological analysis. ExD methods have been used to differentiate the presence of the isoaspartate (isoAsp) from the aspartate (Asp) in peptides and proteins. IsoAsp is a β₃-type amino acid that has an additional methylene group in the backbone, forming a C_α–C_β bond within the polypeptide chain. Cleavage of this bond provides specific fragments that allow differentiation of the isomers. The presence of a C_α–C_β bond within the backbone is unique to β-amino acids, suggesting a similar application of ExD toward the analysis of peptides containing other β-type amino acids. In the current study, ECD and ETD analysis of several β-amino acid containing peptides was performed. It was found that N–C_β and C_α–C_β bond cleavages were rare, providing few c and z type fragments, which was attributed to the instability of the C_β radical. Instead, the electron capture resulted primarily in the formation of a and y fragments, representing an alternative fragmentation pathway, likely initiated by the electron capture at a backbone amide nitrogen protonation site within the β amino acid residues.     

Negative ion electrospray mass spectra of caerulein peptides: an aid to structural determination

MS/MS data derived from the [M-H](-) ions of desulfated caerulein peptides provide (i) sequencing information from a combination of alpha, beta and gamma backbone cleavages, and (ii) identification of specific amino acid side chains by side-chain cleavages [e.g. Ser (-CH(2)O), Thr (-CH(3)CHO) and Asp (-H(2)O)] (fragmentations having no counterparts in positive ion spectra). In addition, delta and/or gamma backbone cleavage ions from Asp residues identify the position of these residues in the peptide. In contrast, neither delta nor gamma cleavage ions are observed from either the Gln2 residue nor from Phe residues. Full structural information can be obtained from a consideration of the positive and negative ion MS/MS data in concert.     

Metastable Atom-Activated Dissociation Mass Spectrometry of Phosphorylated and Sulfonated Peptides in Negative Ion Mode

The dissociation behavior of phosphorylated and sulfonated peptide anions was explored using metastable atom-activated dissociation mass spectrometry (MAD-MS) and collision-induced dissociation (CID). A beam of high kinetic energy helium (He) metastable atoms was exposed to isolated phosphorylated and sulfonated peptides in the 3– and 2– charge states. Unlike CID, where phosphate losses are dominant, the major dissociation channels observed using MAD were C_α – C peptide backbone cleavages and neutral losses of CO₂, H₂O, and [CO₂ + H₂O] from the charge reduced (oxidized) product ion, consistent with an electron detachment dissociation (EDD) mechanism such as Penning ionization. Regardless of charge state or modification, MAD provides ample backbone cleavages with little modification loss, which allows for unambiguous PTM site determination. The relative abundance of certain fragment ions in MAD is also demonstrated to be somewhat sensitive to the number and location of deprotonation sites, with backbone cleavage somewhat favored adjacent to deprotonated sites like aspartic acid residues. MAD provides a complementary dissociation technique to CID, ECD, ETD, and EDD for peptide sequencing and modification identification. MAD offers the unique ability to analyze highly acidic peptides that contain few to no basic amino acids in either negative or positive ion mode.     

The arginine anomaly: arginine radicals are poor hydrogen atom donors in electron transfer induced dissociations

Arginine amide radicals are generated by femtosecond electron transfer to protonated arginine amide cations in the gas phase. A fraction of the arginine radicals formed (2-amino-5-dihydroguanid-1'-yl-pentanamide, 1H) is stable on the 6.7 micros time scale and is detected after collisional reionization. The main dissociation of 1H is loss of a guanidine molecule from the side chain followed by consecutive dissociations of the 2-aminopentanamid-5-yl radical intermediate. Intramolecular hydrogen atom transfer from the guanidinium group onto the amide group is not observed. These results are explained by ab initio and density functional theory calculations of dissociation and transition state energies. Loss of guanidine from 1H is calculated to require a transition state energy of 68 kJ mol(-)(1), which is substantially lower than that for hydrogen atom migration from the guanidine group. The loss of guanidine competes with the reverse migration of the arginine alpha-hydrogen atom onto the guanidyl radical. RRKM calculations of dissociation kinetics predict the loss of guanidine to account for >95% of 1H dissociations. The anomalous behavior of protonated arginine amide upon electron transfer provides an insight into electron capture and transfer dissociations of peptide cations containing arginine residues as charge carriers. The absence of efficient hydrogen atom transfer from charge-reduced arginine onto sterically proximate amide group blocks one of the current mechanisms for electron capture dissociation. Conversely, charge-reduced guanidine groups in arginine residues may function as radical traps and induce side-chain dissociations. In light of the current findings, backbone dissociations in arginine-containing peptides are predicted to involve excited electronic states and proceed by the amide superbase mechanism that involves electron capture in an amide pi* orbital, which is stabilized by through-space coulomb interaction with the remote charge carriers.     

Backbone cleavages of [M - H](-) anions of peptides. Cyclisation of citropin 1 peptides involving reactions between the C-terminal [CONH](-) residue and backbone amide carbonyl groups. A new type of beta cleavage: a joint experimental and theoretical study

This paper reports the study of backbone cleavages in the collision-induced negative-ion mass spectra of the [M - H](-) anions of some synthetic modifications of the bioactive amphibian peptide citropin 1 (GLFDVIKKVASVIGGL-NH(2)). The peptides chosen for study contain no amino acid residues which could effect facile side-chain cleavage, i.e. Ser (-CH(2)O, side-chain cleavage) and Asp (-H(2)O) are replaced by Ala or Lys. We expected that such peptides should exhibit standard and pronounced peaks due to alpha cleavage ions (and to a lesser extent beta cleavage ions) in their collision-induced negative-ion spectra. This expectation was realised, but the spectra also contained peaks formed by a new series of cleavage anions. These are produced following cyclisation of the C-terminal CONH(-) moiety at carbonyl functions of amide groups along the peptide backbone; effectively transferring the NH of the C-terminal CONH(-) group to other amino acid residues. We have called the product anions of these processes beta' ions, in order to distinguish them from standard beta ions. Some beta' ions also fragment directly to some other beta' ions of smaller mass. The reaction coordinates of alpha,beta and beta' backbone processes have been calculated at the HF/6-31G*//AM1 level theory for simple model systems. The initial cyclisation step of the beta' sequence is barrierless and exothermic. Subsequent steps have a maximum barrier of +40 kcal mol(-1), with the overall reaction being endothermic by some 30 kcal mol(-1) at the level of theory used. These calculations take no account of the complexity of the conformationally flexible peptide system, and it is surprising that each of the two reacting centres can 'find' each other in such a large system.     

Radical Stability Directs Electron Capture and Transfer Dissociation of β‐Amino Acids in Peptides

We report on the characteristics of the radical‐ion‐driven dissociation of a diverse array of β‐amino acids incorporated into α‐peptides, as probed by tandem electron‐capture and electron‐transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β‐amino acids than for their α‐form counterparts. In particular, only aromatic (e.g., β‐Phe), and to a substantially lower extent, carbonyl‐containing (e.g., β‐Glu and β‐Gln) amino acid side chains, lead to N? C_β bond cleavage in the corresponding β‐amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical‐driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α‐amino acids, ECD of peptides composed mainly of β‐amino acids reveals a shift in cleavage priority from the N? C_β to the C_α? C bond. The incorporation of CH₂ groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical‐driven β‐amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.     

Specific photodissociation of peptides with multi-stage mass spectrometry

We report collision-induced dissociation (CID) and laser-induced dissociation (LID) performed at different wavelengths between 220 and 280 nm of the peptides leucine-enkephalin (protonated) and gramicidin A (sodiated). Hydrogen-atom losses and side-chain cleavages were observed in LID experiments. These losses depend on the laser wavelength and lead to the formation of radical ions. The fragmentations of these radicals, which are not observed in CID experiments, were investigated in multi-stage mass spectrometry experiments.