Dissociation Chemistry of Hydrogen-Deficient Radical Peptide Anions

The fragmentation chemistry of anionic deprotonated hydrogen-deficient radical peptides is investigated. Homolytic photodissociation of carbon–iodine bonds with 266 nm light is used to generate the radical species, which are subsequently subjected to collisional activation to induce further dissociation. The charges do not play a central role in the fragmentation chemistry; hence deprotonated peptides that fragment via radical directed dissociation do so via mechanisms which have been reported previously for protonated peptides. However, charge polarity does influence the overall fragmentation of the peptide. For example, the absence of mobile protons favors radical directed dissociation for singly deprotonated peptides. Similarly, a favorable dissociation mechanism initiated at the N-terminus is more notable for anionic peptides where the N-terminus is not protonated (which inhibits the mechanism). In addition, collisional activation of the anionic peptides containing carbon–iodine bonds leads to homolytic cleavage and generation of the radical species, which is not observed for protonated peptides presumably due to competition from lower energy dissociation channels. Finally, for multiply deprotonated radical peptides, electron detachment becomes a competitive channel both during the initial photoactivation and following subsequent collisional activation of the radical. Possible mechanisms that might account for this novel collision-induced electron detachment are discussed.     

Tautomerization and Dissociation of Molecular Peptide Radical Cations

Radical‐mediated dissociations of peptide radical cations have intriguing unimolecular gas phase chemistry, with cleavages of almost every bond of the peptide backbone and amino acid side chains in a competitive and apparently “stochastic” manner. Challenges of unraveling mechanistic details are related to complex tautomerizations prior to dissociations. Recent conjunctions of experimental and theoretical investigations have revealed the existence of non‐interconvertible isobaric tautomers with a variety of radical‐site‐specific initial structures, generated from dissociative electron transfer of ternary metal‐ligand‐peptide complexes. Their reactivity is influenced by the tautomerization barriers, perturbing the nature, location, or number of radical and charge site(s), which also determine the energetics and dynamics of the subsequent radical‐mediated dissociatons. The competitive radical‐ and charge‐induced dissociations are extremely dependent on charge density. Charge sequesting can reduce the charge densities on the peptide backbone and hence enhance the flexibility of structural rearrangement. Analysing the structures of precursors, intermediates and products has led to the discovery of many novel radical migration prior to peptide backbone and/or side chain fragmentations. Upon these successes, scientists will be able to build peptide cationic analogues/tautomers having a variety of well‐defined radical sites.     

Formation and Dissociation of Phosphorylated Peptide Radical Cations

In this study, we generated phosphoserine- and phosphothreonine-containing peptide radical cations through low-energy collision-induced dissociation (CID) of the ternary metal?Cligand phosphorylated peptide complexes [Cu^II(terpy) _p M]^·2+ and [Co^III(salen) _p M]^·+ [ _p M: phosphorylated angiotensin III derivative; terpy: 2,2':6',2''-terpyridine; salen: N,N '-ethylenebis(salicylideneiminato)]. Subsequent CID of the phosphorylated peptide radical cations ( _p M^·+) revealed fascinating gas-phase radical chemistry, yielding (1) charge-directed b- and y-type product ions, (2) radical-driven product ions through cleavages of peptide backbones and side chains, and (3) different degrees of formation of [M ?C H₃PO₄]^·+ species through phosphate ester bond cleavage. The CID spectra of the _p M^·+ species and their non-phosphorylated analogues featured fragment ions of similar sequence, suggesting that the phosphoryl group did not play a significant role in the fragmentation of the peptide backbone or side chain. The extent of neutral H₃PO₄ loss was influenced by the peptide sequence and the initial sites of the charge and radical. A preliminary density functional theory study, at the B3LYP 6-311++G(d,p) level of theory, of the neutral loss of H₃PO₄ from a prototypical model??N-acetylphosphorylserine methylamide??revealed several factors governing the elimination of neutral phosphoryl groups through charge- and radical-induced mechanisms.     

Fragmentation of Singly,Doubly, and Triply Charged Hydrogen Deficient Peptide Radical Cations in Infrared Multiphoton Dissociation and Electron Induced Dissociation

Gas phase fragmentation of hydrogen deficient peptide radical cations continues to be an active area of research. While collision induced dissociation (CID) of singly charged species is widely examined, dissociation channels of singly and multiply charged radical cations in infrared multiphoton dissociation (IRMPD) and electron induced dissociation (EID) have not been, so far, investigated. Here, we report on the gas phase dissociation of singly, doubly and triply charged hydrogen deficient peptide radicals, [M + nH]^(n+1)+· (n = 0, 1, 2), in MS³ IRMPD and EID and compare the observed fragmentation pathways to those obtained in MS³ CID. Backbone fragmentation in MS³ IRMPD and EID was highly dependent on the charge state of the radical precursor ions, whereas amino acid side chain cleavages were largely independent of the charge state selected for fragmentation. Cleavages at aromatic amino acids, either through side chain loss or backbone fragmentation, were significantly enhanced over other dissociation channels. For singly charged species, the MS³ IRMPD and EID spectra were mainly governed by radical-driven dissociation. Fragmentation of doubly and triply charged radical cations proceeded through both radical- and charge-driven processes, resulting in the formation of a wide range of backbone product ions including, a-, b-, c-, y-, x-, and z-type. While similarities existed between MS³ CID, IRMPD, and EID of the same species, several backbone product ions and side chain losses were unique for each activation method. Furthermore, dominant dissociation pathways in each spectrum were dependent on ion activation method, amino acid composition, and charge state selected for fragmentation.     

Dissociation Channel Dependence on Peptide Size Observed in Electron Capture Dissociation of Tryptic Peptides

Electron capture dissociation (ECD) of a series of five residue peptides led to the observation that these small peptides did not lead to the formation of the usual c/z ECD fragments, but to a, b, y, and w fragments. In order to determine how general this behavior is for small sized peptides, the effect of peptide size on ECD fragments using a complete set of ECD spectra from the SwedECD spectra database was examined. Analysis of the database shows that b and w fragments are favored for small peptide sizes and that average fragment size shows a linear relationship to parent peptide size for most fragment types. From these data, it appears that most of the w fragments are not secondary fragments of the major z ions, in sharp contrast with the proposed mechanism leading to these ions. These data also show that c fragment distributions depend strongly on the nature of C-terminal residue basic site: arginine leads to loss of short neutral fragments, whereas lysine leads to loss of longer neutral fragments. It also appears that b ions might be produced by two different mechanisms depending on the parent peptide size. A model for the fragmentation pathways in competition is proposed. These relationships between average fragment size and parent peptide size could be further exploited also for CID fragment spectra and could be included in fragmentation prediction algorithms.     

Effects of Peptide Backbone Amide-to-Ester Bond Substitution on the Cleavage Frequency in Electron Capture Dissociation and Collision-Activated Dissociation

Probing the mechanism of electron capture dissociation on variously modified model peptide polycations has resulted in discovering many ways to prevent or reduce $ {\text{N}} - {{\text{C}}_α } $ {\text{N}} - {{\text{C}}_α } bond fragmentation. Here we report on a rare finding of how to increase the backbone bond dissociation rate. In a number of model peptides, amide-to-ester backbone bond substitution increased the frequency of $ {\text{O}} - {{\text{C}}_α } $ {\text{O}} - {{\text{C}}_α } bond cleavage (an analogue of $ {\text{N}} - {{\text{C}}_α } $ {\text{N}} - {{\text{C}}_α } bonds in normal peptides) by several times, at the expense of reduced frequency of cleavages of the neighboring $ {\text{N}} - {{\text{C}}_α } $ {\text{N}} - {{\text{C}}_α } bonds. In contrast, the ester linkage was only marginally broken in collisional dissociation. These results further highlight the complementarity of the reaction mechanisms in electron capture dissociation (ECD) and collision-activated dissociation (CAD). It is proposed that the effects of amide-to-ester bond substitution on fragmentation are mainly due to the differences in product ion stability (ECD, CAD) as well as proton affinity (CAD). This proposal is substantiated by calculations using density functional theory. The implications of these results in relation to the current understanding of the mechanisms of electron capture dissociation and electron transfer dissociation are discussed.     

Electron Capture Dissociation and Collision Induced Dissociation of S-Dipalmitoylated Peptides

Here we investigate the effect of S-dipalmitoylation on the electron capture dissociation (ECD) behavior of peptides. The ECD and collision induced dissociation (CID) of peptides modified by covalent attachment of [(RS)-2,3-di(palmitoyloxy)-propyl] (PAM2) group to cysteine residues [C(PAM2)LEYDTGFK and RPPGC(PAM2)SPFK] were examined. The results suggest that ECD of S-dipalmitoylated peptides can provide both primary sequence information and structural information regarding the modification. The structural information provided by CID is complementary to that provided by ECD.

Coherent Vibrational and Dissociation Dynamics of Polyatomic Radical Cations

The ultrafast dynamics of polyatomic radical cations contribute to important processes including energy transfer in photovoltaics, electron transfer in photocatalysis, radiation-induced DNA damage, and chemical reactions in the upper atmosphere and space. Probing these dynamics in the gas phase is challenging due to the rapid dissociation of polyatomic radical cations following electron removal, which arises from excess electronic excitation of the molecule during the ionization process. This Concept article introduces the reader to how the pump-probe technique of femtosecond time-resolved mass spectrometry (FTRMS) can overcome this challenge to capture coherent vibrational dynamics on the femtosecond timescale in polyatomic radical cations and enable the analysis of their dissociation pathways. Examples of FTRMS applied to three families of polyatomic radical cations are discussed.     

Electron Capture Dissociation of Trivalent Metal Ion-Peptide Complexes

With electrospray ionization from aqueous solutions, trivalent metal ions readily adduct to small peptides resulting in formation of predominantly (peptide + M_T ? H)²⁺, where M_T = La, Tm, Lu, Sm, Ho, Yb, Pm, Tb, or Eu, for peptides with molecular weights below ~1000 Da, and predominantly (peptide + M_T)³⁺ for larger peptides. ECD of (peptide + M_T ? H)²⁺ results in extensive fragmentation from which nearly complete sequence information can be obtained, even for peptides for which only singly protonated ions are formed in the absence of the metal ions. ECD of these doubly charged complexes containing M_T results in significantly higher electron capture efficiency and sequence coverage than peptide-divalent metal ion complexes that have the same net charge. Formation of salt-bridge structures in which the metal ion coordinates to a carboxylate group are favored even for (peptide + M_T)³⁺. ECD of these latter complexes for large peptides results in electron capture by the protonation site located remotely from the metal ion and predominantly c/z fragments for all metals, except Eu³⁺, which undergoes a one electron reduction and only loss of small neutral molecules and b/y fragments are formed. These results indicate that solvation of the metal ion in these complexes is extensive, which results in the electrochemical properties of these metal ions being similar in both the peptide environment and in bulk water.      

电子捕获裂解质谱研究进展

过去的二十年中，以电子捕获裂解为代表的离子反应技术，极大地推动了气相自由基离子化学的研究.该领域的发展是现代质谱的前沿问题，同时也是后基因组时代生物分析的重要技术手段.总结了电子捕获裂解的研究进展，并对其产生、裂解规律以及机理的研究进行了详细的综述.     

Leveraging Electron Transfer Dissociation for Site Selective Radical Generation: Applications for Peptide Epimer Analysis

Traditional electron-transfer dissociation (ETD) experiments operate through a complex combination of hydrogen abundant and hydrogen deficient fragmentation pathways, yielding c and z ions, side-chain losses, and disulfide bond scission. Herein, a novel dissociation pathway is reported, yielding homolytic cleavage of carbon–iodine bonds via electronic excitation. This observation is very similar to photodissociation experiments where homolytic cleavage of carbon–iodine bonds has been utilized previously, but ETD activation can be performed without addition of a laser to the mass spectrometer. Both loss of iodine and loss of hydrogen iodide are observed, with the abundance of the latter product being greatly enhanced for some peptides after additional collisional activation. These observations suggest a novel ETD fragmentation pathway involving temporary storage of the electron in a charge-reduced arginine side chain. Subsequent collisional activation of the peptide radical produced by loss of HI yields spectra dominated by radical-directed dissociation, which can be usefully employed for identification of peptide isomers, including epimers.

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Graphical Abstract ?

     

Charge Transfer Dissociation (CTD) Mass Spectrometry of Peptide Cations Using Kiloelectronvolt Helium Cations

A kiloelectronvolt beam of helium ions is used to ionize and fragment precursor peptide ions starting in the 1+ charge state. The electron affinity of helium cations (24.6 eV) exceeds the ionization potential of protonated peptides and can therefore be used to abstract an electron from—or charge exchange with—the isolated precursor ions. Kiloelectronvolt energies are used, (1) to overcome the Coulombic repulsion barrier between the cationic reactants, (2) to overcome ion-defocussing effects in the ion trap, and (3) to provide additional activation energy. Charge transfer dissociation (CTD) of the [M+H]⁺ precursor of Substance P gives product ions such as [M+H]^2+? and a dominant series of a ions in both the 1+ and 2+ charge states. These observations, along with the less-abundant a + 1 ions, are consistent with ultraviolet photodissociation (UVPD) results of others and indicate that C–C_α cleavages are possible through charge exchange with helium ions. Although the efficiencies and timescale of CTD are not yet suitable for on-line chromatography, this new approach to ion activation provides an additional potential tool for the interrogation of gas phase ions. Graphical Abstract

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Charge‐Site‐Dependent Dissociation of Hydrogen‐Rich Radical Peptide Cations upon Vacuum UV Photoexcitation

Here, 193 nm vacuum ultraviolet photodissociation (VUVPD) was used to investigate the fragmentation of hydrogen‐rich radical peptide cations generated by electron transfer reactions. VUVPD offers new insight into the factors that drive radical‐ and photon‐directed processes. The location of a basic Arg site influences photon‐activated C_α? C(O) bond cleavages of singly charged peptide radical cations, an outcome attributed to the initial conformation of the peptide as supported by molecular dynamics simulated annealing and the population of excited states upon UV excitation. This hybrid ETD/VUVPD method was employed to identify phosphorylation sites of the kinase domain of human TRPM7/ChaK1.     

Electron Capture,Collision-Induced,and Electron Capture-Collision Induced Dissociation in Q-TOF

Recently, we demonstrated that a radio-frequency-free electromagnetostatic (rf-free EMS) cell could be retrofitted into a triple quad mass spectrometer to allow electron-capture dissociation (ECD) without the aid of cooling gas or phase-specific electron injection into the cell (Voinov et al., Rapid Commun Mass Spectrom 22, 3087–3088, 2008; Voinov et al., Anal Chem 81, 1238–1243, 2009). Subsequently, we used our rf-free EMS cell in the same instrument platform to demonstrate ECD occurring in the same space and at the same time with collision-induced dissociation (CID) to produce golden pairs and even triplets from peptides (Voinov et al., Rapid Commun Mass Spectrom 23, 3028–3030, 2009). In this report, we demonstrate that ECD and CID product-ion mass spectra can be recorded at high resolution with flexible control of fragmentation processes using a newly designed cell installed in a hybrid Q-TOF tandem mass spectrometer.     

Gas-Phase Peptide Sulfinyl Radical Ions: Formation and Unimolecular Dissociation

A variety of peptide sulfinyl radical (RSO?) ions with a well-defined radical site at the cysteine side chain were formed at atmospheric pressure (AP), sampled into a mass spectrometer, and investigated via collision-induced dissociation (CID). The radical ion formation was based on AP reactions between oxidative radicals and peptide ions containing single inter-chain disulfide bond or free thiol group generated from nanoelectrospray ionization (nanoESI). The radical induced reactions allowed large flexibility in forming peptide radical ions independent of ion polarity (protonated or deprotonated) or charge state (singly or multiply charged). More than 20 peptide sulfinyl radical ions in either positive or negative ion mode were subjected to low energy collisional activation on a triple-quadrupole/linear ion trap mass spectrometer. The competition between radical- and charge-directed fragmentation pathways was largely affected by the presence of mobile protons. For peptide sulfinyl radical ions with reduced proton mobility (i.e., singly protonated, containing basic amino acid residues), loss of 62?Da (CH₂SO), a radical-initiated dissociation channel, was dominant. For systems with mobile protons, this channel was suppressed, while charge-directed amide bond cleavages were preferred. The polarity of charge was found to significantly alter the radical-initiated dissociation channels, which might be related to the difference in stability of the product ions in different ion charge polarities.     

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.     

Mechanistic Study on Electron Capture Dissociation of the Oligosaccharide-Mg2+ Complex

Electron capture dissociation (ECD) has shown great potential in structural characterization of glycans. However, our current understanding of the glycan ECD process is inadequate for accurate interpretation of the complex glycan ECD spectra. Here, we present the first comprehensive theoretical investigation on the ECD fragmentation behavior of metal-adducted glycans, using the cellobiose-Mg₂₊ complex as the model system. Molecular dynamics simulation was carried out to determine the typical glycan-Mg₂₊ binding patterns and the lowest-energy conformer identified was used as the initial geometry for density functional theory-based theoretical modeling. It was found that the electron is preferentially captured by Mg₂₊ and the resultant Mg_+? can abstract a hydroxyl group from the glycan moiety to form a carbon radical. Subsequent radical migration and α-cleavage(s) result in the formation of a variety of product ions. The proposed hydroxyl abstraction mechanism correlates well with the major features in the ECD spectrum of the Mg₂₊-adducted cellohexaose. The mechanism presented here also predicts the presence of secondary, radical-induced fragmentation pathways. These secondary fragment ions could be misinterpreted, leading to erroneous structural determination. The present study highlights an urgent need for continuing investigation of the glycan ECD mechanism, which is imperative for successful development of bioinformatics tools that can take advantage of the rich structural information provided by ECD of metal-adducted glycans.

Mechanistic Investigation of Phosphate Ester Bond Cleavages of Glycylphosphoserinyltryptophan Radical Cations under Low-Energy Collision-Induced Dissociation

Under the conditions of low-energy collision-induced dissociation (CID), the canonical glycylphosphoserinyltryptophan radical cation having its radical located on the side chain of the tryptophan residue ([G _p SW]^?+) fragments differently from its tautomer with the radical initially generated on the α-carbon atom of the glycine residue ([G^? _p SW]⁺). The dissociation of [G^? _p SW]⁺ is dominated by the neutral loss of H₃PO₄ (98 Da), with backbone cleavage forming the [b₂ – H]^?+/y₁ ⁺ pair as the minor products. In contrast, for [G _p SW]^?+, competitive cleavages along the peptide backbone, such as the formation of [G _p SW – CO₂]^?+ and the [c₂?+?2H]⁺/[z₁ – H]^?+ pair, significantly suppress the loss of neutral H₃PO₄. In this study, we used density functional theory (DFT) to examine the mechanisms for the tautomerizations of [G^? _p SW]⁺ and [G _p SW]^?+ and their dissociation pathways. Our results suggest that the dissociation reactions of these two peptide radical cations are more efficient than their tautomerizations, as supported by Rice–Ramsperger–Kassel–Marcus (RRKM) modeling. We also propose that the loss of H₃PO₄ from both of these two radical cationic tautomers is preferentially charge-driven, similar to the analogous dissociations of even-electron protonated peptides. The distonic radical cationic character of [G^? _p SW]⁺ results in its charge being more mobile, thereby favoring charge-driven loss of H₃PO₄; in contrast, radical-driven pathways are more competitive during the CID of [G _p SW]^?+.

Radical Cations of Dithia- and Tetraselenaradialenes with Restricted Delocalization of the Unpaired Electron

The radical cations of the permethylated dithia[6]radialene 1 and the tetraselena[8]radialene 2 have been investigated by ESR- and ENDOR-spectroscopy. It is shown that in the radical cation of 1 the unpaired electron is delocalized only in one half of the molecule. The magnitude of the (33)S coupling constant suggests a restricted localization within the 2,3-dithiatetramethyl-butadiene unit. We ascribe the slow electron transfer from one part of the molecule to the other to steric effects. The analysis of the ESR spectrum of 2(*)()(+)() shows that only two of the four selenium atoms are involved. The delocalization of the unpaired electron is restricted to only one of the divinyl diselenide moieties with a slow electron transfer rate to the other one.     

Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation

The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99 %. This nearly complete labeling avoids making complex peptide mixtures even more complex because of partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ~90% of its precursor ions with z? > ?2, compared with less than 40 % for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g., 70 % for modified versus only 43 % for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50 % increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.