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Tracking Radical Migration in Large Hydrogen Deficient Peptides with Covalent Labels: Facile Movement does not Equal Indiscriminate Fragmentation
Abstract
Photodissociation of iodo-tyrosine modified peptides yields localized radicals on the tyrosine side chain, which can be further dissociated by collisional activation. We have performed extensive experiments on model peptides, RGYALG, RGYG, and their derivatives, to elucidate the mechanisms underlying backbone fragmentation at tyrosine. Neither acetylation nor deuteration of the tyrosyl phenolic hydrogen significantly affects backbone fragmentation. However, deuterium migration from the tyrosyl beta carbon is concomitant with cleavage at tyrosine. Substitution of tyrosine with 4-hydroxyphenylglycine, which does not have beta hydrogens, results in almost complete elimination of backbone fragmentation at tyrosine. These results suggest that a radical situated on the beta carbon is required for a-type fragmentation in hydrogen-deficient radical peptides. Replacement of the alphaH of the residue adjacent to tyrosine with methyl groups results in significant diminution of backbone fragmentation. The initial radical abstracts an alphaH from the adjacent amino acid, which is poised to "rebound" and abstract the betaH of tyrosine through a six-membered transition-state. Subsequent beta-scission leads to the observed a-type backbone fragment. These results from deuterated peptides clearly reveal that radical migration in peptides can occur and that multiple migrations are not infrequent. Counterintuitively, close examination of all experimental results reveals that the probability for fragmentation at a particular residue is well correlated with thermodynamic radical stability. A-type fragmentation therefore appears to be most likely when favorable thermodynamics are combined with the relevant kinetic control. These results are consistent with ab initio calculations, which demonstrate that barriers to migration are significantly smaller in magnitude than probable dissociation thresholds.
Supplementary resource (1)
... In contrast, the selective bond cleavage that produces Nterminal [ [23][24][25], although there may be several different sites of the radical depending upon the methods used, such as HE-CID and UVPD. Scheme 1. Conventional mechanism for the formation of [a] + ion in high-energy CID [20,21] and UVPD of peptides [22,23] Scheme 2. Conventional mechanism for the formation of [a] + and [x] + ions in MALDI-ISD with hydrogen-abstracting matrix [32][33][34] Regarding the [a] + ion formation and selective Cα-C bond cleavage, Ly and Julian reported that in PD-CID experiments of aromatic amino acid (AR) containing peptides and proteins, the [a] + ions are produced by the cleavage at the Cα-C bond of AR-Xxx residues via RDD reaction of the [M − Hβ + H] ·+ ions generated with successive radical migration [26,27]. Chu [28,29]. ...
... All calculations were performed using ab initio methods combined with density-functional theory (DFT) restricted/ unrestricted B3LYP [35,36] levels of theory and 6-31++G(d,p) basis set in the Gaussian 09 [37] suite of programs. The initial structures of a model peptide AAA and radical species [AAA − H] · were generated using CS Chem3D Ultra [23-25, 28, 29], PD-CID [26,27], and ETD-CID [30,31] combined with DFT calculations Figure 1, it can be observed that the MALDI-ISD experiments with 4,1-NNL result in specific cleavage at the Cα-C bond of the peptide backbone independent of the charge-sites. In MADLI-ISD experiments of peptides and proteins, it is known that the prompt cleavage occurring within several tens ns in the ion source takes place independent of ionization (protonation/deprotonation) [38,39]. ...
... With respect to the specific cleavage at the Cα-C bond, it is reasonable to assume that a Cαcentered radical peptide [M − Hβ + H] ·+ as shown in Scheme 3 is formed as a favorable radical precursor in the specific and preferential formation of the N-terminal [a] + ions and Cterminal [x] + , [y + 2H] + , [y] + , and [w] + ions. As is described in a later subsection, the RDD reactions in Scheme 3 based on the mechanism by Cα-centered radical ions [M − Hβ + H] ·+ [23][24][25][26][27][28][29][30][31] can explain the fact that [a] + ions arising from the cleavage at Cα-C bond of Gly-Xxx residues are missing from the ISD spectra. Furthermore, the formation of [b] + , [y] + , and ...
Radical-driven dissociation (RDD) of hydrogen-deficient peptide ions [M − H + H]·+ has been examined using matrix-assisted laser dissociation/ionization in-source decay mass spectrometry (MALDI-ISD MS) with the hydrogen-abstracting matrices 4-nitro-1-naphthol (4,1-NNL) and 5-nitrosalicylic acid (5-NSA). The preferential fragment ions observed in the ISD spectra include N-terminal [a] + ions and C-terminal [x]+, [y + 2]+, and [w]+ ions which imply that β-carbon (Cβ)-centered radical peptide ions [M − Hβ + H]·+ are predominantly produced in MALDI conditions. RDD reactions from the peptide ions [M − Hβ + H]·+ successfully explains the fact that both [a]+ and [x]+ ions arising from cleavage at the Cα-C bond of the backbone of Gly-Xxx residues are missing from the ISD spectra. Furthermore, the formation of [a]+ ions originating from the cleavage of Cα-C bond of deuterated Ala(d3)-Xxx residues indicates that the [a]+ ions are produced from the peptide ions [M − Hβ + H]·+ generated by deuteron-abstraction from Ala(d3) residues. It is suggested that from the standpoint of hydrogen abstraction via direct interactions between the nitro group of matrix and hydrogen of peptides, the generation of the peptide radical ions [M − Hβ + H]·+ is more favorable than that of the α-carbon (Cα)-centered radical ions [M − Hα + H]·+ and the amide nitrogen-centered radical ions [M − HN + H]·+, while ab initio calculations indicate that the formation of [M − Hα + H]·+ is energetically most favorable.
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... Dissociation of odd-electron peptide radical ions provides additional sequence information that is complementary to that obtained from the dissociation of even-electron protonated peptides. A remarkable range of unimolecular gas-phase chemistry results from dissociations of peptide radical ions occurring at the N-C a [17][18][19][20] or C a -C bonds [21][22][23] of the peptide backbones as well as at the bonds of the amino acid side chains (e. g., C a -C b or C b -C g bonds). [23][24][25][26][27][28][29] The products of the side chain bond cleavages can provide invaluable information allowing identification of structural isomers (e. g., leucine and isoleucine residues) [30,31] that are isobaric and non-differentiable based only on the products of backbone dissociations. ...
... In contrast, charge mobility is hindered in basic residue-containing M * + species, thereby favoring radical-driven fragmentations. The radicaldriven fragmentations lead to N-C a ,N-terminal amide, and C a -C bond cleavages to form [c n + 2H] + /[z n -H] * + ions, [17][18][19][20] y n-1 + ions, [24,25,29] and a n + /[z n + H] * + ions, [21][22][23] respectively, that differ from charge-induced amide bond cleavages. Known side chain reactions include C a -C b and C b -C g bond cleavages; [23][24][25][26][27][28][29] they can be used as structural signatures of the individual amino acid residues in the peptide sequence. ...
... Similar to the N-C a bond cleavage, the cleavage of an C a -C bond of an M * + species is also a radical-driven process involving a b-radical tautomer with the unpaired electron located at the bond-cleaving residue. [22,56,60,66,138,150,151] Systematic experimental and theoretical examinations of novel N-terminal C a -C bond cleavages of a series of tyrosine (a non-basic residue)-containing peptides have revealed that this interesting dissociation pathway involves a p-centered radical tautomer with the unpaired electron and charge located on the aromatic side chain of the tyrosine residue, forming [x n-1 + H] * + ions. [124] A direct N-terminal C a -C bond cleavage produces an ion-molecule complex in which a protonated imine from the N-terminus is solvated by the C-terminal C= O radical. ...
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.
... Major interests of generation of large peptide/protein radical cations have been continuously growing since the success of electron capture dissociation 1 and electron transfer dissociation, 2 which involve recombination of a multiply protonated peptide/protein ion ([M+nH] n+ ) with a low-energy electron, resulting in hydrogen-rich peptide radical cations ([M+nH] •(n-1)+ ). Other techniques, for example collisioninduced oxidative dissociations of transition metal -peptide complexes, [3][4][5] photo-induced dissociations of iodo-peptides, 6,7 and free radical initiated peptide sequencing, 8 have also been developed to generate peptide radical cations that are stoichiometrically equivalent to [M] •+ . ...
... 17,21,[24][25][26][27] Competition between the cleavages of the N-C ␣ and C ␣ -C bonds apparently depends on the properties of the amino acid residues 17,21,28 and the availability of a freely mobile proton. 5,7,29,30 However, their exact roles on these competitive bond cleavages were not studied in detail. ...
... 42,43 Cleavages of the N-C ␣ or C ␣ -C bonds result in c/z-type or a/x-type fragments, respectively. 5,7,17,21,[28][29][30] Scheme 1A illustrates the homolytic cleavages of a neutral -radical tautomer of [Ac(Ala)NHMe -H] • . This structure mimics a peptide radical in which the charge (proton) is sequestered by a basic residue that is far away from the -radical center. ...
Selective cleavages of N-Cα and Cα-C bonds of β-radical tautomers of amino acid residues in radical peptides have been examined theoretically by means of the density functional theory at the M06-2X/6-311++G(d,p) level. The majority of the bond cleavages are homolytic via β-scission. Their energy barriers depend largely on the ability of the radical being stabilized in the transition structures and the availability of a mobile proton in the vicinity of the β-radical center. The N-Cα bond is less favorably cleaved than the Cα-C bond (except Ser and Thr) for systems without a mobile proton. It is because, firstly, the homolytic cleavage is less favorable for the more polar N-Cα bond than for the less polar Cα-C bond. Secondly, a less stable σ-radical localized on the amide nitrogen atom of the incipient N-terminal fragment is formed for the former, while a more stable radical delocalized in a π∗(CO)-like orbital of the incipient C-terminal fragment is formed for the latter. In the presence of a mobile proton N-terminal to the β-radical center, some degrees of heterolytic cleavage character, as preferred by the polar N-Cα bond, are observed. Consequently, its barrier is reduced. If the mobile proton is located at the C-terminal amide oxygen of the β-radical center, the Cα-C bond cleavage will be significantly suppressed. It is because the radical in the incipient C-terminal fragment becomes more localized as a σ-radical on the carbon atom of its protonated amide group. With basic amino acid residues, the Cα-C bond cleavage can be reactivated. Heterolytic cleavage of the polar N-Cα bond can be largely facilitated if a mobile proton N-terminal to the β-radical center is available and the radical in the incipient C-terminal fragment is sufficiently stabilized, for instance, by the aromatic side chain of Trp and Tyr. Therefore, cleavages of the N-Cα bond induced by the β-radical tautomer of Trp and Tyr are often preferred as compared with cleavages of the Cα-C bond in peptide radical cations containing mobile protons.
... In addition, high energy collisions with molecular oxygen of protonated amino acids [3], multiply protonated peptides [4], and multiply proton-ated lysozyme (n=7-17) [5], and also femtosecond laserinduced ionization/dissociation of protonated peptides [6] can be used for the generation of hydrogen deficient species. Other methods are based on chemical derivatization of peptides or proteins to introduce a labile bond, which can then be homolytically cleaved in CID or photodissociation to produce hydrogen deficient molecular ions [7][8][9][10][11][12][13]. Recently, a new method, based on corona discharge initiated electrochemical ionization, has been shown to generate hydrogen deficient species from even electron precursor ions complexed with Fe of the stainless steel spraying tip or with Cu(II) added to the analyte solution [14]. ...
... In addition to the investigations focusing on the generation of hydrogen deficient peptide radical cations, several reports have focused on their gas phase dissociation, mainly in CID [11,21,[27][28][29][32][33][34][35][36][37][38][39][40][41][42]. In the initial report of Siu and coworkers it has been demonstrated that CID of hydrogen deficient species exhibit different fragmentation patterns when compared to those obtained in CID of the even electron species [15]. ...
... Radical migration is another important factor affecting the fragmentation of large hydrogen deficient species as demonstrated by Julian and coworkers [11]. Radical migration was also reported in CID of peptide radical cations of DRVG · IHPF for which bond cleavages were remote from the initial location of the radical site suggesting that for this peptide the radical site is rather mobile [35]. ...
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 MS^3 IRMPD and EID and compare the observed fragmentation pathways to those obtained in MS^3 CID. Backbone fragmentation in MS^3 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 MS^3 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.
... Collisional activation (CA) of the transition metal-ligand ternary complexes, [M n+ (L) m− (P)] (n − m)+ , was developed by Siu and colleagues to introduce a radical site in peptides, where M is a transition metal, L is a ligand, and P is a peptide [25][26][27][28][29][30][31]. Alternatively, UV photolysis of the iodine-containing compound was mainly explored by the Julian group [32,33]. Photodetachment of anionic peptides offers another effective method of introducing a radical in peptides [34,35]. ...
... Once a radical site is generated on a peptide, the unique radical-initiated peptide-backbone dissociation process ensues usually via thermal heating by CA. So far, extensive experimental and theoretical studies have enhanced the understanding of the radical's stability as well as that of radical transfer and radical-initiated dissociation mechanisms [28,29,[31][32][33][38][39][40][41]. ...
Free radical–initiated peptide sequencing mass spectrometry (FRIPS MS) was employed to analyze a number of representative singly or doubly protonated phosphopeptides (phosphoserine and phosphotyrosine peptides) in positive ion mode. In contrast to collision-activated dissociation (CAD) results, a loss of a phosphate group occurred to a limited degree for both phosphoserine and phosphotyrosine peptides, and thus, localization of a phosphorylated site was readily achieved. Considering that FRIPS MS supplies a substantial amount of collisional energy to peptides, this result was quite unexpected because a labile phosphate group was conserved. Analysis of the resulting peptide fragments revealed the extensive production of a-, c-, x-, and z-type fragments (with some minor b- and y-type fragments), suggesting that radical-driven peptide fragmentation was the primary mechanism involved in the FRIPS MS of phosphopeptides. Results of this study clearly indicate that FRIPS MS is a promising tool for the characterization of post-translational modifications such as phosphorylation.
... • L] + , and likewise for other combinations of ␣ and  radical positions. It is recognized that specifying the complete sequence of a peptide fragment is not always convenient, especially when the peptide is long, and that the use of [M−106] •+ to signify loss of the tyrosine sidechain and [M−129] •+ to indicate loss of the tryptophan side-chain has had wide adoption [12,[22][23][24][25]. The use of this shorthand notation is not discouraged as long as the identity of the residue involved and the location of the radical are clear. ...
... Similarly, Wee et al. [28] used G • XR and GX • R to indicate that the radical is located on the first and second residue, respectively; inclusion of a subscript to indicate the position of the radical (e.g., G ␣ • XR and GX ␣ • R) can further enhance the clarity, and is encouraged where appropriate. It is also noteworthy that this nomenclature system is applicable to the products of radical-driven peptide fragmentation mass spectrometry in which a radical initiator is conjugated to some part of the peptide [25,29,30]. We recommend adoption of the all-explicit system in describing the fragmentation of anionic peptides, which exhibits extensive product diversity and complexity, e.g., in the dissociation of the ...
The multitude of fragmentation techniques available to modern tandem mass spectrometry introduces diversity in the types of product ions. An all-explicit nomenclature system for the product ions of peptide fragmentation is herein proposed for the sake of clarity and unambiguity. All variables - the charge, radical and hydrogens gained or lost - associated with a given product ion are specified.
... 35,37 Similarly, the β-radical can also induce cleavages of C α −C bonds to result in the formation of a-type ions. 38,39 Statistical studies have demonstrated that cleavage of the C α −C bond C-terminal to the benzylic-like β-radical isomer is preferable. 26 The complementary x-type ions resulting from the C α −C bond cleavages are normally not observed because they are unstable against further dissociation to form the z-type ions. ...
... Very interestingly, an uncommonly observed x-type ion was produced, namely, [x 2 + H] •+ (m/z 266), which presumably formed through cleavage of the C α −C bond at the asparagine residue together with a hydrogen atom transfer from the N-terminus to the C-terminal fragment. 26,38,39 Formation of the present [x n−1 + H] •+ species has previously been observed in the CID of some arginine-containing radical peptide cations, but it has seemed to occur in a stochastic manner. 9,26 In this study, we found that systematically varying the N-terminal residue of [XYG] •+ to an aliphatic amino acid (i.e., X = A, V, L, or I) gave increasingly more abundant [x 2 + H] •+ species (Table 1) Figure S1. ...
Fascinating N-terminal Cα-C bond cleavages in a series of non-basic tyrosine-containing peptide radical cations have been observed under low-energy collision-induced dissociation (CID), leading to the generation of rarely-observed x-type radical fragments, with significant abundances. CID experiments of the radical cations of the alanyltyrosylglycine tripeptide and its analogs suggested that the N-terminal Cα-C bond cleavage, yielding its [x2 + H](•+) radical cation, does not involve an N-terminal α-carbon-centered radical. Theoretical examination of a prototypical radical cation of the alanyltyrosine dipeptide, using density functional theory calculations, suggested that direct N-terminal Cα-C bond cleavage could produce an ion-molecule complex formed between the incipient a1(+) and x1(•) fragments. Subsequent proton transfer from the iminium nitrogen atom in a1(+) to the acyl carbon atom in x1(•) results in the observable [x1 + H](•+). The barriers against this novel Cα-C bond cleavage and the competitive N-Cα bond cleavage, forming the complementary [c1 + 2H](+) / [z1 - H](•+) ion pair, are similar (ca. 16 kcal mol(-1)). Rice-Ramsperger-Kassel-Marcus modeling revealed that [x1 + H](•+) and [c1 + 2H](+) species are formed with comparable rates, in agreement with energy-resolved CID experiments for [AY](•+).
... In order to explore the structural features that lead to these favorable kinetics in greater detail, it is necessary to determine the exact migration route. It has been well established previously that immediately prior to formation of the a 3 ion, the radical must migrate to the tyrosine β carbon as shown in Scheme 1c [15,27]. However, the radical could arrive at the tyrosine β carbon via direct migration or through a multistep route. ...
... Successive radical migration from the Ala α carbon to the Tyr β carbon is expected to be a facile process through a transition state with a six-membered ring. Our previous work has shown that Tyr β radical can migrate from the carbon α position at an adjacent glycine residue [27]. Similarly, the Ala α radical should also be able to migrate to adjacent Tyr β carbon (Scheme 1b). ...
One of the keys for understanding radical directed dissociation in peptides is a detailed knowledge of the factors that mediate radical migration. Peptide radicals can be created by a variety of means; however, in most circumstances, the originally created radicals must migrate to alternate locations in order to facilitate fragmentation such as backbone cleavage or side chain loss. The kinetics of radical migration are examined herein by comparing results from ortho-, meta-, and para-benzoyl radical positional isomers for several peptides. Isomers of a constrained cyclic peptide generated by several orthogonal radical initiators are also probed as a function of charge state. Cumulatively, the results suggest that small changes in radical position can significantly impact radical migration, and overall structural flexibility of the peptide is also an important controlling factor. A particularly interesting pathway for the peptide RGYALG that is sensitive to ortho versus meta or para substitution was fully mapped out by a suite of deuterium labeled peptides. This data was then used to optimize parameters in molecular dynamics-based simulations, which were subsequently used to obtain further insight into the structural underpinnings that most strongly influence the kinetics of radical migration.
... – H or c ● ) compared with those of the even electron precursor ions, reflecting the hydrogendeficient nature of the precursor ions. The exact location of the unpaired electron of the hydrogen-deficient species cannot be unambiguously determined because radical migration has been shown to commonly occur in large hydrogendeficient peptides [13, 20, 24]. However, based on the observation that all product ions containing a Trp were detected with a mass decrement of 1 Da, we can postulate that through radical rearrangements, the unpaired electron of the hydrogen-deficient species migrated to the Trp residue. ...
... Regardless of the precise mechanism(s) by which ECD proceeds, electron capture by the hydrogen-deficient precursor ions will result in two radical sites on the polypeptide. If we further take into consideration that radical migration easily occurs in peptide radicals [20, 24] we can hypothesize that after the initial electron capture event, radical recombination occurs. Such a process will create an even electron species, instead of a radical intermediate, and quenches or suppresses N–C α bond cleavages. ...
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–Cα 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–Cα 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–C_α bond cleavages. Alternatively, radical recombination and N–C_α bond cleavages can be in competition, with radical recombination being the dominant pathway and N–C_α cleavages occurring to a lesser degree. Formation of b- and y-type ions observed for two of the hydrogendeficient peptides examined is also discussed.
... Protein structure can be investigated by monitoring the degree of migration from a specific radical initiation point [16]. The fragmentation pathways and chemistry of positively charged hydrogendeficient radicals have been studied extensively [17][18][19][20][21][22][23][24]. Hydrogen-deficient radical anions have been neglected by comparison [25,26]. ...
... Figure 4a plots the average relative abundance of backbone dissociation for each amino acid. Also shown are the BDEs for the β-hydrogens of each amino acid side chain, which is where backbone dissociation in RDD is typically initiated [17]. As can be seen from the data, lower BDEs (on average) correlate with increased yield of backbone dissociation. ...
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.
... Once created, odd-electron chemistry has many uses in the gas phase, including cross-linking [8], examining three-dimensional structure [9], accessing novel dissociation pathways [10,11], and isomer identification [12]. Radical-directed dissociation (RDD) is often sensitive to fine structural details because dissociation is preceded by radical migration, which is guided by the relative orientation of hydrogen atom(s) that must be abstracted to allow relocation of the nascent radical to the ultimate site of fragmentation [13]. ...
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. Graphical Abstract ?.
... I n the past decade or so, radical-driven peptide fragmentation mass spectrometry (MS) has been a subject extensively investigated by many groups worldwide because of its promising potential as another powerful tool for peptide sequencing tandem mass spectrometry [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. The radical-driven fragmentation MS methods have been studied in a variety of aspects, such as the generation and migration of a radical site [3-5, 8, 11, 17], the stability of radical ions [18,19], peptide fragmentation pathways [20][21][22][23], thermodynamics [6,24], applications [25][26][27], and gas-phase structure elucidations [14,28,29]. Several interesting methodologies have been introduced for generating a radical site on the peptide manifold. ...
The present study demonstrates that one-step peptide backbone fragmentations can be achieved using the TEMPO [2-(2,2,6,6-tetramethyl piperidine-1-oxyl)]-assisted free radical-initiated peptide sequencing (FRIPS) mass spectrometry in a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer and a Q-Exactive Orbitrap instrument in positive ion mode, in contrast to two-step peptide fragmentation in an ion-trap mass spectrometer (reference Anal. Chem. 85, 7044–7051 (30)). In the hybrid Q-TOF and Q-Exactive instruments, higher collisional energies can be applied to the target peptides, compared with the low collisional energies applied by the ion-trap instrument. The higher energy deposition and the additional multiple collisions in the collision cell in both instruments appear to result in one-step peptide backbone dissociations in positive ion mode. This new finding clearly demonstrates that the TEMPO-assisted FRIPS approach is a very useful tool in peptide mass spectrometry research. Graphical Abstractᅟ
... Ion-ion reactions can be used to assign charge states in very large systems [10]. It has been demonstrated that radical fragmentation can yield information about probable close contacts [11][12][13][14][15][16][17]. Various forms of spectroscopy can also be used to obtain information on biomolecules [18,19]. ...
Significant effort is being employed to utilize the inherent speed and sensitivity of mass spectrometry for rapid structural determination of proteins; however, a thorough understanding of factors influencing the transition from solution to gas phase is critical for correct interpretation of the results from such experiments. It was previously shown that combined use of action excitation energy transfer (EET) and simulated annealing can reveal detailed structural information about gaseous peptide ions. Herein, we utilize this method to study microsolvation of charged groups by retention of 18-crown-6 (18C6) in the gas phase. In the case of GTP (CEGNVRVSRE LAGHTGY), solvation of the 2+ charge state leads to reduced EET, whereas the opposite result is obtained for the 3+ ion. For the mini-protein C-Trpcage, solvation by 18C6 leads to dramatic increase in EET for the 3+ ion. Examination of structural details probed by molecular dynamics calculations illustrate that solvation by 18C6 alleviates the tendency of charged side chains to seek intramolecular solvation, potentially preserving native-like structures in the gas phase. These results suggest that microsolvation may be an important tool for facilitating examination of native-like protein structures in gas phase experiments. Graphical Abstractᅟ
... [29] Alternatively, UV-induced photo-activation of gaseous peptide molecular ions can be applied to specifically initiate homolytic cleavage of covalent carbon-iodine bonds, e.g. in aromatic amino acids, such as 3-iodotyrosine, to initially form aryl-centered peptide radicals. [30,31] Porter and Masterson reported the pH-controlled selectivity of aryl peroxycarbonate reagents for peptide derivatization, differentiating between the N-terminus or lysine side chain amino groups, due to the pK a -differences between α-amino versus ε-amino groups. [32] The respective t-butyl peroxycarbamate peptide derivatives then decompose site-specifically upon collision activation and therefore present an elegant access to free radical-promoted peptide cleavage reactions. ...
We have synthesized a homobifunctional active ester cross-linking reagent containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) moiety connected to a benzyl group (Bz), termed TEMPO-Bz-linker. The aim for designing this novel cross-linker was to facilitate MS analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). The TEMPO-Bz-linker was reacted with all 20 proteinogenic amino acids as well as with model peptides to gain detailed insights into its fragmentation mechanism upon collision activation. The final goal of this proof-of-principle study was to evaluate the potential of the TEMPO-Bz-linker for chemical cross-linking studies to derive 3D-structure information of proteins. Our studies were motivated by the well documented instability of the central NO―C bond of TEMPO-Bz reagents upon collision activation. The fragmentation of this specific bond was investigated in respect to charge states and amino acid composition of a large set of precursor ions resulting in the identification of two distinct fragmentation pathways. Molecular ions with highly basic residues are able to keep the charge carriers located, i.e. protons or sodium cations, and consequently decompose via a homolytic cleavage of the NO―C bond of the TEMPO-Bz-linker. This leads to the formation of complementary open-shell peptide radical cations, while precursor ions that are protonated at the TEMPO-Bz-linker itself exhibit a charge-driven formation of even-electron product ions upon collision activation. MS3 product ion experiments provided amino acid sequence information and allowed determining the cross-linking site. Our study fully characterizes the CID behavior of the TEMPO-Bz-linker and demonstrates its potential, but also its limitations for chemical cross-linking applications utilizing the special features of open-shell peptide ions on the basis of selective tandem MS analysis.
... Photodissociation is then used to generate a radical by homolytic cleavage of a photolabile carbon-iodine bond. The initially formed radicals are reactive, but not well placed to induce dissociation [17]. Thus migration of the radical species to locations where dissociation is favorable occurs rapidly [18]. ...
Evaluating protein structure in the gas phase is useful for understanding the intrinsic forces which influence protein folding and for determining the feasibility of probing condensed phase structure with gas phase interrogation. KIX is a three-helix bundle protein that has been reported previously to preserve the condensed phase structure in the gas phase. Herein, structure dependent radical directed dissociation (RDD) is used to examine the gas phase structure of KIX by establishing residue specific distance constraints which can be used to assess candidate structures obtained from molecular dynamics simulations. The data obtained by RDD is consistent with KIX structures that largely retain condensed phase structure as determined previously by NMR. There are several factors that favor retention of the KIX native fold in the gas phase. The structure is largely comprised of alpha helices, which are known to be stable in the gas phase. This is particularly true if the C-terminus of the helix is capped with a positive charge, which occurs for the two most stable helices in KIX. There are several arginine based salt bridges which link critical portions of KIX together. KIX also has an abundance of basic residues; this multiplicity increases the chance that sites which require little structural reorganization following desolvation can be charge carriers. Thus under appropriate conditions, solution phase structure can be largely retained and meaningfully examined in the gas phase.
... Dissociation of the hydrogen rich radical cations often gives rise to c and z@BULLET ions, along with side-chain losses, through which they can convert quickly to hydrogen deficient radical ions192021. Hydrogen deficient cations can be generated by a variety of methods: laser ablation followed by ultraviolet (UV) photoionization [22, 23]; collision-induced dissociation (CID) of metal-ligand-peptide complexes242526; CID of peptide derivatives with labile bonds such as S-nitrosylation [27, 28], serine/homoserine nitrate esters [29], peroxycarbamates [30], 2,2,6,6-tetramethylpiperidine-1-oxy (TEMPO) [31, 32], and 4,4'-azobis(4-cyanopentanoic acid) (Vazo 68) [33] ; UV photolysis of iodinated tyrosine containing peptides [34] ; or noncovalent complexes with photolabile precursor [35] ; electroninduced dissociation of multiply charged ions [36, 37]. Hydrogen deficient radical anions can be formed by electron detachment [38] or photodetachment [39] from multiply deprotonated molecules, CID of peptide–metal complexes [40, 41], and photodissociation of iodinated peptide [42]. ...
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 (CH(2)SO), 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.
... Such mobility was initially hypothesized based on theoretical calculations [15] and was used to explain some fragmentation schemes in peptide mass spectrometry. [23,24] Conversion between SC and CC radicals was even detected in peptides, [25,26] but this is, to our knowledge, the first observation of a real migration of CC radicals. ...
Radical migration between aliphatic amino acid side chains can occur in solution and intramolecularly in peptides. The kinetic constant of the hydrogen transfer reaction was measured by using competition kinetics, and the half-life as well as the distance that a radical can move within a protein was calculated.
We report experimental and computational studies of protonated adenine C-8 σ-radicals that are presumed yet elusive reactive intermediates of oxidative damage to nucleic acids. The radicals were generated in the gas phase by the collision-induced dissociation of C-8-Br and C-8-I bonds in protonated 8-bromo- and 8-iodoadenine as well as by 8-bromo- and 8-iodo-9-methyladenine. Protonation by electrospray of 8-bromo- and 8-iodoadenine was shown by cyclic-ion mobility mass spectrometry (c-IMS) to form the N-1-H, N-9-H and N-3-H, N-7-H protomers in 85:15 and 81:19 ratios, respectively, in accordance with the equilibrium populations of these protomers in water-solvated ions that were calculated by density functional theory (DFT). Protonation of 8-halogenated 9-methyladenines yielded single N-1-H protomers, which was consistent with their thermodynamic stability. The radicals produced from the 8-bromo and 8-iodo adenine cations were characterized by UV-vis photodissociation action spectroscopy (UVPD) and c-IMS. UVPD revealed the formation of C-8 σ-radicals along with N-3-H, N-7-H-adenine π-radicals that arose as secondary products by hydrogen atom migrations. The isomers were identified by matching their action spectra against the calculated vibronic absorption spectra. Deuterium isotope effects were found to slow the isomerization and increase the population of C-8 σ-radicals. The adenine cation radicals were separated by c-IMS and identified by their collision cross sections, which were measured relative to the canonical N-9-H adenine cation radical that was cogenerated in situ as an internal standard. Ab initio CCSD(T)/CBS calculations of isomer energies showed that the adenine C-8 σ-radicals were local energy minima with relative energies at 76-79 kJ mol-1 above that of the canonical adenine cation radical. Rice-Ramsperger-Kassel-Marcus calculations of unimolecular rate constants for hydrogen and deuterium migrations resulting in exergonic isomerizations showed kinetic shifts of 10-17 kJ mol-1, stabilizing the C-8 σ-radicals. C-8 σ-radicals derived from N-1-protonated 9-methyladenine were also thermodynamically unstable and readily isomerized upon formation.
Ion-ion reactions are valuable tools in mass-spectrometry-based peptide and protein sequencing. To boost the generation of sequence-informative fragment ions from low charge-density precursors, supplemental activation methods, via vibrational and photoactivation, have become widely adopted. However, long-lived radical peptide cations undergo intramolecular hydrogen atom transfer from c-type ions to z•-type ions. Here we investigate the degree of hydrogen transfer for thousands of unique peptide cations where electron transfer dissociation (ETD) was performed and was followed by beam-type collisional activation (EThcD), resonant collisional activation (ETcaD), or concurrent infrared photoirradiation (AI-ETD). We report on the precursor charge density and the local amino acid environment surrounding bond cleavage to illustrate the effects of intramolecular hydrogen atom transfer for various precursor ions. Over 30% of fragments from EThcD spectra comprise distorted isotopic distributions, whereas over 20% of fragments from ETcaD have distorted distributions and less than 15% of fragments derived from ETD and AI-ETD reveal distorted isotopic distributions. Both ETcaD and EThcD give a relatively high degree of hydrogen migration, especially when D, G, N, S, and T residues were directly C-terminal to the cleavage site. Whereas all postactivation methods boost the number of c- and z•-type fragment ions detected, the collision-based approaches produce higher rates of hydrogen migration, yielding fewer spectral identifications when only c- and z•-type ions are considered. Understanding hydrogen rearrangement between c- and z•-type ions will facilitate better spectral interpretation. © 2021 American Society for Mass Spectrometry. Published by American Chemical Society. All rights reserved.
Fragmentation of peptide radical cations [M].+ has been examined using matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) with hydrogen-abstracting nitro-substituted matrices. The ISD spectra of peptides containing an arginine (Arg) residue at carboxyl (C)-termini showed preferential [w]+ ions when 4-nitro-1-naphthol (4,1-NNL) matrix was used, whereas the use of 3,5-dinitrosalicylic acid (3,5-DNSA) resulted in preferential [x]+ ions. Minor or some [d]+ , [x]+ , [y]+ , and [z]+ ions were also observed. For peptides containing Arg residue at amino (N)-termini, the ISD spectra showed preferential [a]+ ions independent of matrix used. The observed [a]+ , [w]+ , [x]+ , [y]+ , and [z]+ ions can be rationally explained by radical-directed dissociation (RDD) of the peptide radical cations [M].+ , although [d]+ ions may be formed via Norrish Type I cleavage and/or by RDD of [M].+ ions. The formation of overdegraded [d]+ , [w]+ , [y]+ , and [z]+ ions is discussed from the standpoint of the internal energy of radical cations [M].+ and radical fragment ions [a + H].+ and [x + H].+ deposited via collisional interactions with excited matrix molecules in the MALDI plume. The radical site of the peptide cations [M].+ was presumed to be backbone amide nitrogen, from MALDI-ISD data with three different deuterated amino acids.
The formation and radical-directed dissociation of multiple hydrogen-abstracted peptide cations [M + H - mH]·+ has been reported using MALDI-ISD with dinitro-substituted matrices. The MALDI-ISD of synthetic peptides using 3,5-dinitrosalicylic acid (3,5-DNSA) and 3,4-dinitrobenzoic acid (3,4-DNBA) as matrices resulted in multiple hydrogen abstraction from the analyte [M + H]+ and fragment [a]+ ions, i.e., [M + H - mH]+ and [a - mH]+ (m = 1-8). All of the ISD spectra showed unusually intense [a]+ ions originating from cleavage at the Cα-C bond of the Leu-Xxx residues when peptides without Phe/Tyr/His/Cys residues were used. The intensity of the [a
n
]+ series ions generated using 3,5-DNSA and 3,4-DNBA rapidly decreased with increasing residue number n, suggesting cleavage at multiradical sites of [M + H - mH]•+. It was suggested that multiple hydrogen abstraction from protonated peptides [M + H]+ mainly takes place from the backbone amide nitrogen.
The development of new ion-activation/dissociation methods continues to be one of the most active areas of mass spectrometry owing to the broad applications of tandem mass spectrometry in the identification and structural characterization of molecules. This Review will showcase the impact of ultraviolet photodissociation (UVPD) as a frontier strategy for generating informative fragmentation patterns of ions, especially for biological molecules whose complicated structures, subtle modifications, and large sizes often impede molecular characterization. UVPD energizes ions via absorption of high-energy photons, which allows access to new dissociation pathways relative to more conventional ion-activation methods. Applications of UVPD for the analysis of peptides, proteins, lipids, and other classes of biologically relevant molecules are emphasized in this Review.
Residue-specific cleavages resulting in the formation of [a]⁺ ions of peptides have been examined using 20 keV high-energy (high-E) collision-induced dissociation (CID) with a tandem time-of-flight (TOF/TOF) instrument. High-E CID resulted in relatively sensitive cleavage at the Cα-C bond of β-substituted aliphatic Val/Ile-Xxx and aromatic Phe/Tyr/His-Xxx residues when an Arg residue was located on the N-terminal side. The sensitive aromatic Phe/Tyr/His residues and insensitive Gly residue in high-E CID are similar to other methods, such as ultraviolet photodissociation (UVPD) of [M+H]⁺, low-E CID of [M].+ and MALDI-ISD with a hydrogen-abstracting matrix. The properties of the Val/Ile residues are not necessarily common to the same methods. The high-sensitivity of Val/Ile and Phe/Tyr/His residues to high-E CID may be due to high-energy single collision between target gas and the bulky sidechains of these residues. It is suggested that from high-E CID and MALDI-ISD experiments the formation of [a]⁺ ions is essentially connected with the loss of atomic hydrogen from protonated peptides [M+H]⁺, and that high-E CID results in the loss of hydrogen from the backbone amide nitrogen rather than the site of the β-carbon of sidechains.
The molecular structures of six open-shell z3-ions resulting from electron transfer dissociation mass spectrometry (ETD MS) were investigated using infrared ion spectroscopy in the 800-1850 and 3200-3700 cm-1 spectral range in combination with density functional theory and molecular mechanics/molecular dynamics calculations. We assess in particular the question of whether the radical remains at the C-site of the backbone cleavage, or whether it migrates by H-atom transfer to another, energetically more favorable position. Calculations performed herein as well as by others show that radical migration to an amino acid side chain or to an -carbon along the peptide backbone can lead to structures that more stable, up to 33 kJ/mol for the systems investigated here, by virtue of resonance stabilization of the radical in these alternative positions. Nonetheless, for four out of the six z3-ions considered here, our results quite clearly indicate that radical migration does not occur, suggesting that the radical is kinetically trapped at the site of ETD cleavage. For the two remaining systems, a structural assignment is less secure and we suggest that a mixture of migrated and unmigrated structures may be formed.
Four isomers of the radical cation of tripeptide phenylalanylglycyltryptophan, in which the initial location of the radical center is well defined, have been isolated and their collision-induced dissociation (CID) spectra examined. These ions, the π-centered [FGWπ•]⁺, α-carbon- [FGα•W]⁺, N-centered [FGWN•]⁺ and ζ-carbon- [Fζ•GW]⁺ radical cations, were generated via collision-induced dissociation (CID) of transition metal-ligand-peptide complexes, side chain fragmentation of a π-centered radical cation, homolytic cleavage of a labile nitrogen–nitrogen single bond, and laser induced dissociation of iodinated peptide, respectively. The π-centered and tryptophan N-centered peptide radical cations produced almost identical CID spectra, despite the different locations of their initial radical sites, which indicated that interconversion between the π-centered and tryptophan N-centered radical cations is facile. By contrast, the α-carbon-glycyl radical [FGα•W]⁺, and ζ-phenyl radical [Fζ•GW]⁺, featured different dissociation product ions, suggesting that the interconversions among α-carbon, π-centered (or tryptophan N-centered) and ζ-carbon- radical cations have higher barriers than those to dissociation. Density functional theory calculations have been used to perform systematic mechanistic investigations on the interconversions between these isomers and to study selected fragmentation pathways for these isomeric peptide radical cations. The results showed that the energy barrier for interconversion between [FGWπ•]⁺ and [FGWN•]⁺ is only 31.1 kcal mol⁻¹, much lower than the barriers to their dissociation (40.3 kcal mol⁻¹). For the [FGWπ•]⁺, [FGα•W]⁺, and [Fζ•GW]⁺, the barriers to interconversion are higher than those to dissociation, suggesting that interconversions among these isomers are not competitive with dissociations. The [z3 – H]•+ ions isolated from [FGα•W]⁺ and [Fζ•GW]⁺ show distinctly different fragmentation patterns, indicating that the structures of these ions are different and this result is supported by the DFT calculations.
Macrocyclization is commonly observed in large bn(+) (n ≥ 4) ions and as a consequence can lead to incorrect protein identification due to sequence scrambling. In this work, the analogous [b5 - H]˙(+) radical cations derived from aliphatic hexapeptides (GA5˙(+)) also showed evidence of macrocyclization under CID conditions. However, the major fragmentation for [b5 - H]˙(+) ions is the loss of CO2 and not CO loss, which is commonly observed in closed-shell bn(+) ions. Isotopic labeling using CD3 and (18)O revealed that more than one common structure underwent dissociations. Theoretical studies found that the loss of CO2 is radical-driven and is facilitated by the radical being located at the Cα atom immediately adjacent to the oxazolone ring. Comparable energy barriers against macrocyclization, hydrogen-atom transfer, and fragmentations are found by DFT calculations and the results are consistent with the experimental observations that a variety of dissociation products are observed in the CID spectra.
Peptide radical cations that contain an aromatic amino acid residue cleave to give [zn – H]•+ ions with [b2 – H – 17]•+ and [c1 – 17]+ ions, the dominant products in the dissociation of [zn – H]•+, also present in lower abundance in the CID spectra. Isotopic labeling in the aromatic ring of [Yπ•GG]+ establishes that in the formation of [b2 – H – 17]•+ ions a hydrogen from the δ-position of the Y residue is lost, indicating that nucleophilic substitution on the aromatic ring has occurred. A preliminary DFT investigation of nine plausible structures for the [c1 – 17]+ ion derived from [Yπ•GG]+ shows that two structures resulting from attack on the aromatic ring by oxygen and nitrogen atoms from the peptide backbone have significantly better energies than other isomers. A detailed study of [Yπ•GG]+ using two density functionals, B3LYP and M06-2X, with a 6-31++G(d,p) basis set gives a higher barrier for attack on the aromatic ring of the [zn – H]•+ ion by nitrogen than by the carbonyl oxygen. However, subsequent rearrangements involving proton transfers are much higher in energy for the oxygen-substituted isomer leading to the conclusion that the [c1 – 17]+ ions are the products of nucleophilic attack by nitrogen, protonated 2,7-dihydroxyquinoline ions. The [b2 – H – 17]•+ ions are formed by loss of glycine from the same intermediates involved in the formation of the [c1 – 17]+ ions.
In this installment, we discuss radical ion chemistry as an increasingly important area of mass spectrometry (MS) development and the application to bioanalysis. At the current stage, most of the research is performed by a small set of academic groups. Given the unique capability offered by radical MS compared to the traditional studies on even-electron ions of analytes, it is likely that these types of fundamental studies will attract more attention and even be commercialized in the near future.
Surface-induced dissociation (SID) is a valuable tool for investigating the activation and dissociation of large ions in tandem mass spectrometry. This account summarizes key findings from studies of the energetics and mechanisms of complex ion dissociation in which SID experiments were combined with Rice-Ramsperger-Kassel-Marcus modeling of the experimental data. These studies used time- and collision-energy-resolved SID experiments and SID combined with resonant ejection of selected fragment ions on a specially designed Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Fast-ion activation by collision with a surface combined with the long and variable timescale of FT-ICR mass spectrometry is perfectly suited to studying the energetics and dynamics of complex ion dissociation in the gas phase. Modeling of time- and collision-energy-resolved SID enables the accurate determination of energy and entropy effects in the dissociation process. It has been demonstrated that entropy effects play an important role in determining the dissociation rates of both covalent and noncovalent bonds in large gaseous ions. SID studies have provided important insights on the competition between charge-directed and charge-remote fragmentation in even-electron peptide ions and the role of the charge and radical site on the energetics of the dissociation of odd-electron peptide ions. Furthermore, this work examined factors that affect the strength of noncovalent binding, as well as the competition between covalent and noncovalent bond cleavages and between proton and electron transfer in model systems. Finally, SID studies have been used to understand the factors affecting nucleation and growth of clusters in solution and in the gas phase.
The effects of hydrogen bonding and spin density at the oxygen atom on the gas-phase reactivity of phenoxyl radicals were investigated experimentally and theoretically in model systems and the dipeptide LysTyr. Gas-phase ion-molecule reactions were carried out between radical cations of several aromatic nitrogen bases with the neutrals nitric oxide and n-propyl thiol. Reactivity of radical cations 4–6 correlated with the spin density. The possibility of hydrogen bonding was explored in compounds which allowed four-, five-, and six-membered ring to be formed between the protonated nitrogen and the phenoxyl oxygen, while possessing similar spin density at the oxygen atom. The N+-H⋯O bond length was calculated to decrease in the series (1–3), consistent with the theoretical calculations finding weak hydrogen bonding in 2 and strong hydrogen bonding in 3. This coincided with the decrease in reaction rates of 1–3 with both nitric oxide and n-propyl thiol. DFT calculations found that the lowest energy structure of the distonic radical cation of the dipeptide [LysTyr(O)]+ has a short hydrogen bond between the protonated Lys side chain and the phenoxyl oxygen, 1.70 Å, which is consistent with its low reactivity.
Predominant loss of neutral CO2 has been observed under conditions of low-energy collision-induced dissociation from a prototypical molecular radical cation of the tripeptide aspartylglycylarginine ([DGR]+). The decarboxylation occurs mainly from the side chain of the aspartic acid residue and partially from the C-terminal carboxyl group. The structural and mechanistic features that facilitate CO2 loss from the Asp side chain of [DGR]+ and its chemically modified analogues incorporating methylation have been elucidated using a combination of Rice–Ramsperger–Kassel–Marcus modeling and density functional theory at the B3LYP/6-31 + +G(d,p) level. Current mechanistic investigations suggest that the loss of CO2 from the side chain of the aspartic acid residue involves hydrogen atom transfer from its carboxyl oxygen atom in conjunction with α-centered radical transfer to the β-centered radical on the aspartic acid side chain. Minor CO2 loss from the C-terminal carboxyl group occurs through the [DGαR]+ isomer, with the radical migrating to the α-carbon of the middle Gly residue. Barriers against the CO2 loss from the side chain of the aspartic acid residue and from the C-terminus of [DGαR]+ are approximately 30 and 36 kcal mol−1, respectively.
The collision-induced dissociation (CID) of [b5 – H]•+ ions containing four alanine residues and one tryptophan give identical spectra regardless of the initial location of the tryptophan indicating that, as proposed for b5+ ions, sequence scrambling occurs prior to dissociation. Cleavage occurs predominantly at the peptide bonds and at the N−Cα bond of the alanine residue that is attached to the N-terminus of the tryptophan residue. The product of the latter pathway, an ion at m/z 240, is the base peak in all the mass spectra. With the exception of one minor channel giving a b3+ ion, the product ions retain both the tryptophan residue and the radical. Experiments with one trideuterated alanine established the sequences of loss of alanine residues. Formation of identical products implies a common intermediate, a [b5 – H]•+ ion that has a `linear` structure in which the tryptophan residue is present as an α-radical located in the oxazolone ring, structure Ie. Density Functional Theory calculations show this structure to be at the global minimum, 14.6 kcal mol-1 below the macrocyclic structure, ion II. Loss of CO from the [b5 – H]•+ ions is inhibited by the presence of the radical centre in the oxazolone ring and migration of the proton from the oxazolone ring onto the peptide backbone induces cleavage of an N−Cα or peptide bond. Three calculated structures for the ion at m/z 240 all have an oxazolone ring. Two of these structures may be formed from Ie , depending upon which proton migrates onto the peptide chain prior to the dissociation. The barrier to interconversion between these two structures requires a 1,3-hydrogen atom shift and is high (51.0 kcal mol-1), but both can convert into a third isomer that readily loses CO2 (barrier 38.7 kcal mol-1). The lowest barrier to the loss of CO, the usual fragmentation path observed for protonated oxazolones, is 47.0 kcal mol-1.
The fragmentation products of the ε-carbon-centered radical cations [Y(ε)˙LG](+) and [Y(ε)˙GL](+), made by 266 nm laser photolysis of protonated 3-iodotyrosine-containing peptides, are substantially different from those of their π-centered isomers [Y(π)˙LG](+) and [Y(π)˙GL](+), made by dissociative electron transfer from ternary metal-ligand-peptide complexes. For leucine-containing peptides the major pathway for the ε-carbon-centered radical cations is loss of the side chain of the leucine residue forming [YG(α)˙G](+) and [YGG(α)˙](+), whereas for the π-radicals it is the side chain of the tyrosine residue that is lost, giving [G(α)˙LG](+) and [G(α)˙GL](+). The fragmentations of the product ions [YG(α)˙G](+) and [YGG(α)˙](+) are compared with those of the isomeric [Y(ε)˙GG](+) and [Y(π)˙GG](+) ions. The collision-induced spectra of ions [Y(ε)˙GG](+) and [YGG(α)˙](+) are identical, showing that interconversion occurs prior to dissociation. For ions [Y(ε)˙GG](+), [Y(π)˙GG](+) and [YG(α)˙G](+) the dissociation products are all distinctly different, indicating that dissociation occurs more readily than isomerization. Density functional theory calculations at B3LYP/6-31++G(d,p) gave the relative enthalpies (in kcal mol(-1) at 0 K) of the five isomers to be [Y(ε)˙GG](+) 0, [Y(π)˙GG](+) -23.7, [YGG(α)˙](+) -28.7, [YG(α)˙G](+) -31.0 and [Y(α)˙GG](+) -38.5. Migration of an α-C-H atom from the terminal glycine residue to the ε-carbon-centered radical in the tyrosine residue, a 1-11 hydrogen atom shift, has a low barrier, 15.5 kcal mol(-1) above [Y(ε)˙GG](+). By comparison, isomerization of [Y(ε)˙GG](+) to [YG(α)˙G](+) by a 1-8 hydrogen atom migration from the α-C-H atom of the central glycine residue has a much higher barrier (50.6 kcal mol(-1)); similarly conversion of [Y(ε)˙GG](+) into [Y(π)˙GG](+) has a higher energy (24.4 kcal mol(-1)).
A gas-phase radical rearrangement through intramolecular hydrogen-atom transfer (HAT) was studied in the glutathione radical cation, [γ-ECG]+., which was generated by a homolytic cleavage of the protonated S-nitrosoglutathione. Ion–molecule reactions suggested that the radical migrates from the original sulfur position to one of the α-carbon atoms. Experiments on the radical cations of dipeptides derived from the glutathione sequence, [γ-EC]+. and [CG]+., pointed to the glutamic acid α-carbon atom as the most likely site of the radical migration. Infrared multiple-photon dissociation (IRMPD) spectroscopy was employed to generate complementary information. IRMPD of [γ-ECG]+. in the approximately 1000–1800 cm−1 region was inconclusive owing to the relatively broad, overlapping absorption bands. However, the IRMPD spectrum of [γ-EC]+. in this region was consistent with the radical migrating from the sulfur to the α-carbon atom of glutamic acid. IRMPD in the 2800–3700 cm−1 region performed on [γ-ECG]+. is consistent with a mixture of both the original sulfur-based radical and the resulting glutamic acid α-carbon-based species. Comparisons are made with previously published condensed and gas-phase studies on intramolecular HAT in glutathione.
In recent years, a number of novel tandem mass spectrometry approaches utilizing radical-driven peptide gas-phase fragmentation chemistry have been developed. These approaches show a peptide fragmentation pattern quite different from that of collision-induced dissociation (CID). The peptide fragmentation features of these approaches share some in common with electron capture dissociation (ECD) or electron transfer dissociation (ETD) without the use of sophisticated equipment such as a Fourier-transform mass spectrometer. For example, Siu and coworkers showed that CID of transition metal (ligand)-peptide ternary complexes led to the formation of peptide radical ions through dissociative electron transfer (Chu et al., 2000. J Phys Chem B 104:3393-3397). The subsequent collisional activation of the generated radical ions resulted in a number of characteristic product ions, including a, c, x, z-type fragments and notable side-chain losses. Another example is the free radical initiated peptide sequencing (FRIPS) approach, in which Porter et al. and Beauchamp et al. independently introduced a free radical initiator to the primary amine group of the lysine side chain or N-terminus of peptides (Masterson et al., 2004. J Am Chem Soc 126:720-721; Hodyss et al., 2005 J Am Chem Soc 127: 12436-12437). Photodetachment of gaseous multiply charged peptide anions (Joly et al., 2008. J Am Chem Soc 130:13832-13833) and UV photodissociation of photolabile radical precursors including a C-I bond (Ly & Julian, 2008. J Am Chem Soc 130:351-358; Ly & Julian, 2009. J Am Soc Mass Spectrom 20:1148-1158) also provide another route to generate radical ions. In this review, we provide a brief summary of recent results obtained through the radical-driven peptide backbone dissociation tandem mass spectrometry approach. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 9999:1-17, 2014.
Peptide radical cations A(n)Y•+ (where n = 3, 4 or 5) and A5W•+ have been generated by collision-induced dissociation (CID) of [CuII(tpy)(peptide)]•2+ complexes. Apart from the charge-driven fragmentation at the N-Cα bond of the hetero residue producing either [c + 2H]+ or [z - H]•+ ions and radical-driven fragmentation at the Cα-C bond to give a+ ions, unusual product ions [x + H]•+ and [z + H]•+ are abundant in the CID spectra of the peptides with the hetero residue in the second or third position of the chain. The formation of these ions requires that both the charge and radical be located on the peptide backbone. Energy-resolved spectra established that the [z + H]•+ ion can be produced either directly from the peptide radical cation or via the fragment ion [x + H]•+. Additionally, backbone dissociation by loss of the C-terminal amino acid giving [b(n-1) - H]•+ increases in abundance with the length of the peptides. Mechanisms by which peptide radical cations dissociate have been modeled using density functional theory (B3LYP/6-31++G** level) on tetrapeptides AYAG•+, AAYG•+ and AWAG•+
Free radical-initiated peptide sequencing (FRIPS) mass spectrometry derives advantage from the introduction of highly selective low-energy dissociation pathways in target peptides. An acetyl radical, formed at the peptide N-terminus via collisional activation and subsequent dissociation of a covalently attached radical precursor, abstracts a hydrogen atom from diverse sites on the peptide, yielding sequence information through backbone cleavage as well as side-chain loss. Unique free radical-initiated dissociation pathways observed at serine and threonine residues lead to cleavage of the neighboring N-terminal Cα-C or N-Cα bond rather than the typical Cα-C bond cleavage observed with other amino acids. These reactions were investigated by FRIPS of model peptides of the form AARAAAXAA, where X is the amino acid of interest. In combination with density functional theory (DFT) calculations, the experiments indicate the strong influence of hydrogen bonding at serine or threonine on the observed free radical chemistry. Hydrogen bonding of the side chain hydroxyl group with a backbone carbonyl oxygen aligns the singly-occupied π orbital on the β-carbon and the N-Cα bond, leading to low-barier β-cleavage of the N-Cα bond. Interaction with the N-terminal carbonyl favors a hydrogen-atom transfer process to yield stable c and z(•) ions, while C-terminal interaction leads to effective cleavage of the Cα-C bond through rapid loss of isocyanic acid. Dissociation of the Cα-C bond may also occur via water loss followed by β-cleavage from a nitrogen-centered radical. These competitive dissociation pathways from a single residue illustrate the sensitivity of gas-phase free radical chemistry to subtle factors such as hydrogen bonding that affect the potential energy surface for these low-barrier processes.
Radical-directed dissociation of gas phase ions is emerging as a powerful and complementary alternative to traditional tandem mass spectrometric techniques for biomolecular structural analysis. Previous studies have identified that coupling of 2-[(2,2,6,6-tetramethylpiperidin-1-oxyl)methyl]benzoic acid (TEMPO-Bz) to the N-terminus of a peptide introduces a labile oxygen-carbon bond that can be selectively activated upon collisional activation to produce a radical ion. Here we demonstrate that structurally-defined peptide radical ions can also be generated upon UV laser photodissociation of the same TEMPO-Bz derivatives in a linear ion-trap mass spectrometer. When subjected to further mass spectrometric analyses, the radical ions formed by a single laser pulse undergo identical dissociations as those formed by collisional activation of the same precursor ion, and can thus be used to derive molecular structure. Mapping the initial radical formation process as a function of photon energy by photodissociation action spectroscopy reveals that photoproduct formation is selective but occurs only in modest yield across the wavelength range (300-220 nm), with the photoproduct yield maximised between 235 and 225 nm. Based on the analysis of a set of model compounds, structural modifications to the TEMPO-Bz derivative are suggested to optimise radical photoproduct yield. Future development of such probes offers the advantage of increased sensitivity and selectivity for radical-directed dissociation.
Radical cations [Met-Gly]•+, [Gly-Met]•+, and [Met-Met]•+ have been generated through collision-induced dissociation (CID) of [CuII(CH3CN)2(peptide)]•2+ complexes. Their fragmentation patterns and dissociation mechanisms have been studied both experimentally and theoretically using density functional theory at the UB3LYP/6-311++G(d,p) level. The captodative structure, in which the radical is located at the α-carbon of the N-terminal residue and the proton is on the amide oxygen, is the lowest energy structure on each potential energy surface. The canonical structure, with the charge and spin both located on the sulfur, and the distonic ion with the proton on the terminal amino group, and the radical on the α-carbon of the C-terminal residue have similar energies. Interconversion between the canonical structures and the captodative isomers is facile and occurs prior to fragmentation. However, isomerization to produce the distonic structure is energetically less favorable and cannot compete with dissociation except in the case of [Gly-Met]•+. Charge-driven dissociations result in formation of [b
n
– H]•+ and a
1
ions. Radical-driven dissociation leads to the loss of the side chain of methionine as CH3-S-CH = CH2 producing α-glycyl radicals from both [Gly-Met]•+ and [Met-Met]•+. For [Met-Met]•+, loss of the side chain occurs at the C-terminal as shown by both labeling experiments and computations. The product, the distonic ion of [Met-Gly]•+, NH3+CH(CH2CH2SCH3)CONHCH•COOH dissociates by loss of CH3S•. The isomeric distonic ion NH3+CH2CONHC•(CH2CH2SCH3)COOH is accessible directly from the canonical [Gly-Met]•+ ion. A fragmentation pathway that characterizes this ion (and the distonic ion of [Met-Met]•+) is homolytic fission of the Cβ–Cγ bond to lose CH3SCH2•.
Time- and collision energy-resolved surface-induced dissociation (SID) of peptide radical anions was studied for the first time using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) configured for SID experiments. Peptide radical cations and anions were produced by gas-phase fragmentation of CoIII(salen)-peptide complexes. The effect of the charge, radical, and the presence of a basic residue on the energetics and dynamics of dissociation of peptide ions was examined using RVYIHPF (1) and HVYIHPF (2) as model systems. Comparison of the survival curves for of [M+H]{sup +}, [M-H]â», M{sup +{sm_bullet}}, and [M-2H]{sup -{sm_bullet}} ions of these precursors demonstrated that even-electron ions are more stable towards fragmentation than their odd-electron counterparts. RRKM modeling of the experimental data demonstrated that the lower stability of the positive radicals is mainly attributed to lower dissociation thresholds while entropy effects are responsible the relative instability of the negative radicals. Substitution of arginine with less basic histidine residue has a strong destabilizing effect on the [M+H]{sup +} ions and a measurable stabilizing effect on the odd-electron ions. Lower threshold energies for dissociation of both positive and negative radicals of 1 are attributed to the presence of lower-energy dissociation pathways that are most likely promoted by the presence of the basic residue.
We report electron-transfer dissociation (ETD) mass spectra of histidine-containing peptides DSHAK, FHEK, HHGYK, and HHSHR from trypsinolysis of histatin 5. ETD of both doubly and triply protonated peptides provided sequence ions of the c and z type. In addition, electron transfer to doubly protonated peptides produced abundant long-lived cation-radicals, (M+2H)+, whose relative intensities depended on the peptide sequence and number of histidine residues. CID-MS3 spectra of (M+2H)+ cation-radicals were entirely different from the ETD spectra of the doubly charged ions and involved radical-driven losses of C4H6N2 neutral fragments from the histidine residues and charge-driven backbone cleavages forming b and y ions. Product ions from CID of (M+2H)+ were further characterized by CID-MS4 spectra to distinguish the histidine residues undergoing loss of C4H6N2. The ETD-CID-MSn mass spectra are interpreted by considering radical-induced rearrangements of histidine side chains in the long-lived charge-reduced ions.
Two types of aminoketyl radicals and cation-radicals are distinguished by each's structure and reactivity. As proper aminoketyl radicals we denote the presumed intermediates of NCα bond dissociations induced by electron attachment to protonated peptides. These radicals have pyramidized aminoketyl groups, CαC(OH)NH, high spin density on the central carbon atom, and undergo facile NCα bond dissociations. The critical energies for NCα bond cleavages in proper aminoketyl radicals are summarized here and typically do not exceed 60kJmol−1 in peptide cation-radicals. In contrast, a different type of intermediates, which we call improper aminoketyl radicals, is formed by collisional dissociation of peptide cation-radicals, such as decarboxylation of zn fragments. Improper aminoketyl radicals have near planar CαC(OH)NH groups, and the spin density is delocalized over several atoms adjacent to the aminoketyl moiety. Improper aminoketyl radicals show higher transition energies for NCα bond cleavage and undergo H-atom transfers resulting in side-chain losses.
Peptide dissociation behavior in TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)-based FRIPS (free radical initiated peptide sequencing) mass spectrometry was analyzed in both positive- and negative-ion modes for a number of standard peptides including angiotensin II, kinetensin, glycoprotein IIb fragment (296-306), des-Pro2-bradykinin, and ubiquitin tryptic fragment (43-48). In the positive mode, the •Bz-C(O)-peptide radical species was produced exclusively at the initial collisional activation of o-TEMPO-Bz-C(O)-peptides, and two consecutive applications of collisional activation were needed to observe peptide-backbone fragments. In contrast, in the negative-ion mode, a single application of collisional activation to o-TEMPO-Bz-C(O)-peptides produced extensive peptide-backbone fragmentations as well as •Bz-C(O)-peptide radical species. This result indicates that the duty-cycle in the TEMPO-based FRIPS mass spectrometry can be reduced by one-half in the negative-ion mode. In addition, the fragment ions observed in the negative-ion experiments were mainly of the a-, c-, x-, and z-types, indicating that radical-driven tandem mass spectrometry was mainly responsible for the TEMPO-based FRIPS even with a single application of collisional activation. Furthermore, the survival fraction analysis of o-TEMPO-Bz-C(O)-peptides was made as a function of the applied normalized collision energy (NCE). This helped us to better understand the differences in FRIPS behavior between the positive- and negative-ion modes in terms of dissociation energetics. The duty-cycle improvement made in the present study provides a cornerstone for future research aiming to achieve a single-step FRIPS in the positive-ion mode.
The salient aspect of cation radical reactions is that they are mostly radical driven. They involve omolytic bond cleavages and hydrogen atom migrations. Mass spectrometry of small free radicals has been reviewed. Dissociations of even-electron ions under slow heating conditions are dominated by heterolytic bond cleavages accompanied by proton or larger group migrations that often result in quite complicated reaction pathways. In the particular case of peptide even-electron ions, the main dissociations are eliminations of small molecules (water, ammonia) and proton-driven cleavages of amide bonds. The latter are essential for peptide sequencing by mass spectrometry. A different approach to peptide cation radicals relied on transition metal complexes produced by electrospray that showed radical-driven dissociations such as homolytic bond dissociations upon collisional activation in the slow heating regime. With a proper choice of the metal ion and organic ligands, peptide ternary complexes have been shown to undergo intramolecular electron transfer upon collisional activation, producing metal-free peptide ions.
We report the implementation and evaluation of activated ion negative electron transfer dissociation (AI-NETD) in order to enhance the analytical capabilities of NETD for the elucidation of doubly deprotonated peptide anions. The analytical figures-of-merit and fragmentation characteristics are compared for NETD alone and with supplemental collisional activation of the charge reduced precursors or infrared photoactivation of the entire ion population during the NETD reaction period. The addition of supplemental collisional activation of charge reduced precursor ions or infrared photoactivation of the entire ion population concomitant with the NETD reaction period significantly improves sequencing capabilities for peptide anions as evidenced by the greater abundances of product ions and overall sequence coverage. Neither of these two AI-NETD methods significantly alters the net fragmentation efficiencies relative to NETD; however, the sequence ion conversion percentages with respect to formation of diagnostic product ions are notably higher. Supplemental infrared photoactivation outperforms collisional activation for most of the peptide fragmentation metrics evaluated.
The fragmentation behavior of various cysteine sulfinyl ions (intact, N-acetylated, and O-methylated), a new member of the gas-phase amino acid radical ion family, was investigated by low energy collision-induced dissociation (CID). The dominant fragmentation channel for the protonated cysteine sulfinyl radicals (SO•Cys) was the radical-directed Cα-Cβ homolytic cleavage, resulting in the formation of glycyl radical ions and a loss of CH2SO. This channel, however, was not observed for protonated N-acetyl cysteine sulfinyl radical (Ac-SO•Cys); instead, a charge-directed water loss followed by an immediate SH loss prevailed. Counter-intuitively, the water loss did not derive from the carboxylic acid, but involved the sulfinyl oxygen, a proton, and a Cβ-hydrogen atom. Theoretical calculations suggested that N-acetylation significantly increased the energy barrier (~14 kcal/mol) for the radical-directed fragmentation channel due to its reduced capability in stabilizing the thus formed glycyl radical ions via captodative effect. N-acetylation also assisted in mobilizing the proton to the sulfinyl site, which reduced the energy barrier for the H2O loss. Our studies demonstrated that for cysteine sulfinyl radical ions, the stability of the product ions (glycyl radical ions) and the location of charge (proton) could significantly modulate the competition between radical- and charge-directed fragmentation.
Structural investigations of large biomolecules in the gas phase are challenging. Herein, it is reported that action spectroscopy taking advantage of facile carbon-iodine bond dissociation can be used to examine the structures of large molecules, including whole proteins. Iodotyrosine serves as the active chromophore, which yields distinctive spectra depending on the solvation of the side chain by the remainder of the molecule. Isolation of the chromophore yields a double featured peak at ∼290 nm, which becomes a single peak with increasing solvation. Deprotonation of the side chain also leads to reduced apparent intensity and broadening of the action spectrum. The method can be successfully applied to both negatively and positively charged ions in various charge states, although electron detachment becomes a competitive channel for multiply charged anions. In all other cases, loss of iodine is by far the dominant channel which leads to high sensitivity and simple data analysis. The action spectra for iodotyrosine, the iodinated peptides KGYDAKA, DAYLDAG, and the small protein ubiquitin are reported in various charge states.
In this study, we used collision-induced dissociation (CID) to examine the gas-phase fragmentations of [G(n)W](•+) (n = 2-4) and [GXW](•+) (X = C, S, L, F, Y, Q) species. The C(β)-C(γ) bond cleavage of a C-terminal decarboxylated tryptophan residue ([M - CO(2)](•+)) can generate [M - CO(2) - 116](+), [M - CO(2) - 117](•+), and [1H-indole](•+) (m/z 117) species as possible product ions. Competition between the formation of [M - CO(2) - 116](+) and [1H-indole](•+) systems implies the existence of a proton-bound dimer formed between the indole ring and peptide backbone. Formation of such a proton-bound dimer is facile via a protonation of the tryptophan γ-carbon atom as suggested by density functional theory (DFT) calculations. DFT calculations also suggested the initially formed ion 2, the decarboxylated species that is active against C(β)-C(γ) bond cleavage, can efficiently isomerize to form a more stable π-radical isomer (ion 9) as supported by Rice-Ramsperger-Kassel-Marcus (RRKM) modeling. The C(β)-C(γ) bond cleavage of a tryptophan residue also can occur directly from peptide radical cations containing a basic residue. CID of [WG(n)R](•+) (n = 1-3) radical cations consistently resulted in predominant formation of [M - 116](+) product ions. It appears that the basic arginine residue tightly sequesters the proton and allows the charge-remote C(β)-C(γ) bond cleavage to prevail over the charge-directed one. DFT calculations predicted that the barrier for the former is 6.2 kcal mol(-1) lower than that of the latter. Furthermore, the pathway involving a salt-bridge intermediate also was accessible during such a bond cleavage event.
Dissociation of peptide radical ions involves competition between charge-induced and radical-induced reactions that can be preceded by isomerization. The isomeric radical cations of the peptide methyl ester [G˙GR-OMe](+) and [GG˙R-OMe](+) provide very similar collision-induced dissociation (CID) spectra, suggesting that isomerization occurs prior to fragmentation. They undergo characteristic radical-induced bond cleavage of the peptide N-terminal amide bond resulting in the y(2)(+) ion, and of the arginine side-chain's C(α)-C(β) bond giving protonated allylguanidine {[CH(2)[double bond, length as m-dash]CHCH(2)NHC(NH(2))(2)](+), m/z 100}. The absence of a y(2)(+) fragment ion in the CID of the radical cationic tripeptide [A(CH(3))G˙R](+) and of an m/z 100 ion in the spectrum of [G˙A(CH(3))R](+) (where A(CH(3)) is an α-aminoisobutyric acid residue, which cannot form an α-carbon-centered radical through hydrogen atom transfer) establishes the importance of hydrogen atom migration along the peptide backbone prior to specific radical-induced fragmentations. Herein we use density functional theory (DFT) at the B3LYP/6-31++G(d,p) level to evaluate the barriers for interconversion between the α-carbon-centered radicals and for dissociation. The radical cations [G˙GR](+) and [GG˙R](+) have their radicals located on the α-carbon atoms of the peptide backbone and their charge densities largely sequestered on the guanidine groups of the side-chain of arginine residues. This is in contrast to the isomeric radical cations of [GGG]˙(+), in which the charge resides necessarily on the peptide backbone. The lower charge densities on the backbones of [G˙GR](+) and [GG˙R](+) result in greater structural flexibility, decreasing the barrier for interconversion between these α-carbon-centered radicals to 36.2 kcal mol(-1) (cf. 44.7 kcal mol(-1) for [GGG]˙(+)). The total absence of charge, assessed by examining intramolecular hydrogen atom transfers among the three α-carbon centers of the isomeric neutral α-carbon-centered triglycine radicals [GGG-H]˙, leads to an additional but slight reduction in enthalpy, to approximately 34 kcal mol(-1).
In this study, we observed unprecedented cleavages of the Cβ–Cγ bonds of tryptophan residue side chains in a series of hydrogen-deficient tryptophan-containing peptide radical cations (M•+) during low-energy collision-induced dissociation (CID). We used CID experiments and theoretical density functional theory (DFT) calculations to study the mechanism of this bond cleavage, which forms [M – 116]+ ions. The formation of an α-carbon radical intermediate at the tryptophan residue for the subsequent Cβ–Cγ bond cleavage is analogous to that occurring at leucine residues, producing the same product ions; this hypothesis was supported by the identical product ion spectra of [LGGGH – 43]+ and [WGGGH – 116]+, obtained from the CID of [LGGGH]•+ and [WGGGH]•+, respectively. Elimination of the neutral 116-Da radical requires inevitable dehydrogenation of the indole nitrogen atom, leaving the radical centered formally on the indole nitrogen atom ([Ind]•-2), in agreement with the CID data for [WGGGH]•+ and [W1-CH3GGGH]•+; replacing the tryptophan residue with a 1-methyltryptophan residue results in a change of the base peak from that arising from a neutral radical loss (116 Da) to that arising from a molecule loss (131 Da), both originating from Cβ–Cγ bond cleavage. Hydrogen atom transfer or proton transfer to the γ-carbon atom of the tryptophan residue weakens the Cβ–Cγ bond and, therefore, decreases the dissociation energy barrier dramatically.
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The online version of this article (doi:10.1007/s13361-011-0295-5) contains supplementary material, which is available to authorized users.
Electron-capture dissociation (ECD) is a new fragmentation technique that utilizes ion-electron recombination reactions. The latter have parallels in other research fields; revealing these parallels helps to understand the ECD mechanism. An overview is given of ECD-related phenomena and of the history of ECD discovery and development. Current views on the ECD mechanism are discussed using both published and new examples.
The quantum yields of I∗(2P1/2) production from iodobenzene and pentafluoroiodobenzene at five different dissociation wavelengths of 222, 236, 266, 280, and ∼305 nm are presented and compared with those obtained from nonaromatic cyclic iodides (i.e., cyclohexyl iodide and adamantyl iodide). The I(2P3/2) and I∗(2P1/2) atoms generated in the photolysis of the above iodides were monitored using a two-photon laser-induced fluorescence technique. From the measured I∗ quantum yields, two general observations are made for aryl iodides. They are that (i) the I∗ yield is influenced by the σ∗←n as well as π∗←π transitions at all photolysis wavelengths within the A band and (ii) there is a clear indication of a fluorine substitution effect on the dynamics of I∗ production. The contribution from the benzene type π∗←π transition varies with excitation wavelength. Fluorine substitution in aryl iodides is found to increase the I∗ quantum yield similar to what is reported in alkyl iodides. The effect of fluorine substitution is more pronounced at the red edge of the A-band excitation than at any other wavelengths. This is explained by invoking the presence of a charge-transfer band arising due to the transition of a 5pπ nonbonding iodine electron to the π∗ molecular orbital near the red edge of the A band. This charge-transfer state is coupled more strongly to the 3Q1 state of the σ∗←n transition in pentafluoroiodobenzene than in iodobenzene. The dynamics of I∗ formation is found to be unaltered by ring strain in cyclic iodides except at the blue wing excitation. At the blue wing, B-band transitions affect the dynamics of I∗ production in cyclic iodides, leading to the formation of more I∗ from adamantyl iodide. © 2002 American Institute of Physics.
Peptide sequence analysis using a combination of gas-phase ion/ion chemistry and tandem mass spectrometry (MS/MS) is demonstrated. Singly charged anthracene anions transfer an electron to multiply protonated peptides in a radio frequency quadrupole linear ion trap (QLT) and induce fragmentation of the peptide backbone along pathways that are analogous to those observed in electron capture dissociation. Modifications to the QLT that enable this ion/ion chemistry are presented, and automated acquisition of high-quality, single-scan electron transfer dissociation MS/MS spectra of phosphopeptides separated by nanoflow HPLC is described.
• electron capture dissociation
• fragmentation
• ion/ion reactions
• charge transfer
• ion trap
The rapid identification of proteins from biological samples is critical for extracting useful information in proteomics studies. Mass spectrometry is one among the various methods of choice for achieving this task; however, current approaches are limited by a lack of chemical control over proteins in the gas phase. Herein, it is shown that modification of tyrosine to iodo-tyrosine followed by UV photodissociation of the carbon-iodine bond can be used to generate a radial site specifically at the modified residue. The subsequent dissociation of the protein is largely dominated by radical-directed reactions, including dominant backbone fragmentation at the modified tyrosine. If iodination of the protein is carried out under natively folded conditions, the modification and ultimate fragmentation can typically be isolated to a single tyrosine residue. Some secondary backbone cleavage in the immediate vicinity of the modified tyrosine also occurs, especially if proline is present. In the absence of a reactive tyrosine residue, similar chemistry occurs via iodination at histidine. Possible mechanisms which would lead to the observed a-type fragments at tyrosine and the secondary fragments at proline are discussed. A method for using this type of site-specific information to reduce database searching times in proteomics experiments by several orders of magnitude is outlined.
AbstractA linear synchronous transit or quadratic synchronous transit approach is used to get closer to the quadratic region of the transition state and then quasi‐newton or eigenvector following methods are used to complete the optimization. With an empirical estimate of the hessian, these methods converge efficiently for a variety of transition states from a range of starting structures.
We measured absolute rate coefficients for the reactions of the hydroxyl radical with methane (k[sub 1]) and methane-d[sub 4] (k[sub 2]) using the laser photolysis/laser-induced fluorescence technique. We characterized k[sub 1] and k[sub 2] over the temperature range 293-800 K at pressures between 400 and 750 Torr of helium. We find excellent agreement between our results and the recent determinations of k[sub 1] at lower temperatures by Vaghjiani and Ravishankara. The measured rate coefficients, in the units cm[sup 3] molecule[sup [minus]1] s[sup [minus]1], fit well to the three-parameter expressions k[sub 1](T) = 9.65 [times] 10[sup [minus]20] T[sup 2.58] exp(-1082/T) and k[sub 2](T) = 8.70 x 10[sup [minus]22] T[sup 3.23] exp(-1334/T). The kinetic isotope effect for abstraction of the H and D atoms varies from 6.75 at 293 K to 1.96 at 800 K. We compare our results to recently reported calculations by Melissas and Truhlar. 10 refs., 3 figs., 1 tab.
For Abstract see ChemInform Abstract in Full Text.
A new amino-protecting group, the 9-fluorenylmethyloxycarbonyl group (FMOC), which is stable toward acids and catalytic hydrogenation but readily cleaved under mildly basic, nonhydrolytic conditions, is reported. The FMOC group may be introduced by reaction of the amine with 9-fluorenylmethyl chloroformate. A number of protected amino acid derivatives were coupled with other amino acids or esters by use of the corresponding N-hydroxypiperidine esters. Deblocking of the FMOC group was carried out with liquid ammonia or at room temperature with piperidine, morpholine, ethanolamine, etc.
Disulfide bonds in gaseous multiply-protonated proteins are preferentially cleaved in the mass spectrometer by low-energy electrons, in sharp contrast to excitation of the ions by photons or low-energy collisions. For S−S cyclized proteins, capture of one electron can break both an S−S bond and a backbone bond in the same ring, or even both disulfide bonds holding two peptide chains together (e.g., insulin), enhancing the sequence information obtainable by tandem mass spectrometry on proteins in trace amounts. Electron capture at uncharged S−S is unlikely; cleavage appears to be due to the high S−S affinity for H• atoms, consistent with a similar favorability found for tryptophan residues. RRKM calculations indicate that H• capture dissociation of backbone bonds in multiply-charged proteins represents nonergodic behavior, as proposed for the original direct mechanism of electron capture dissociation.
A new hybrid Hartree-Fock-density functional (HF-DF) model called the modified Perdew-Wang 1-parameter model for kinetics (MPW1K) is optimized against a database of 20 forward barrier heights, 20 reverse barrier heights, and 20 energies of reaction. The results are compared to other hybrid HF-DF methods with the 6-31+G(d,p) basis. The new method reduces the mean unsigned error in reaction barrier heights by a factor of 2.4 over MPW1PW91 and by a factor of 3 over B3LYP.
Irradiation of protonated polypeptides NH2–RH+–COOH by >10 eV electrons leads to further ionization and fast intramolecular charge transfer to the free N-terminus. The resulting species may undergo further hydrogen atom rearrangement to form distonic ions N+H3–RH+–COO. Such transfer is exothermic but can involve an appreciable barrier, e.g., 2.3±0.5 eV for MH2+ ions of the peptide ACTH 1–10. Radical polypeptide dications can, therefore, be viewed as hydrogen atom wires. Subsequent capture of low energy electrons results in fragmentation. The pattern of this electronic excitation dissociation (EED) is consistent with hydrogen transfer prior to electron capture.
Intramolecular H-atom transfer in model peptide-type radicals was investigated with high-level quantum-chemistry calculations. Examination of 1,2-, 1,3-, 1,5-, and 1,6[C <-> N]-H shifts, 1,4- and 1,7[C C]-H shifts, and 1,4[N N]-H shifts (Scheme 1), was carried out with a number of theoretical methods. In the first place, the performance of UB3-LYP (with the 6-31G(d), 6-31G(2dfP), and 6-311+G(d,p) basis sets) and UMP2 (with the 6-31G(d) basis set) was assessed for the determination of radical geometries. We found that there is only a small basis-set dependence for the UB3-LYP structures, and geometries optimized with UB3-LYP/6-31G(d) are generally sufficient for use in conjunction with high-level composite methods in the determination of improved H-transfer thermochemistry. Methods assessed in this regard include the high-level composite methods, G3(MP2)-RAD, CBS-QB3, and G3//B3-LYP, as well as the density-functional methods B3-LYP, MPWB1K, and BMK in association with the 6-31+G(dp) and 6-311++G(3df3pd) basis sets. The high-level methods give results that are close to one another, while the recently developed functionals MPWB1K and BMK provide cost-effective alternatives. For the systems considered, the transformation of an N-centered radical to a C-centered radical is always exothermic (by 25 kJ (.) mol' or more), and this can lead to quite modest barrier heights of less than 60 kJ (.) mol(-1) (specifically for 1,5[C N]-H and 1,6[C N]-H shifts). H-Migration barriers appear to decrease as the ring size in the transition structure (TS) increases, with a lowering of the barrier being found, for example when moving from a rearrangement proceeding via a four-membered-ring TS (e.g., the 1,3[C <-> N]-H shift, CH3-C(O)-NH center dot -> center dot CH2-C(O)-NH2) to a rearrangement proceeding via a six-membered-ring TS (e.g., the 1,5[C <-> N]-H shift, (NH)-N-center dot-CH2-C(O)-NH-CH3 <-> NH2-CH2-C(O)-NH-CH2 center dot).
Electrospray ionization (ESI) mass spectrometry of methanolic solutions of mixtures of the copper salt (2,2′:6′,2″-terpyridine)copper(II) nitrate monohydrate ([Cu(II)(tpy)(NO3)2]·H2O) and a tripeptide GXR (where X = 1 of the 20 naturally occurring amino acids) yielded [Cu(II)(tpy)(GXR)]2+ ions, which were then subjected to collision induced dissociation (CID). In all but one case (GRR), these [Cu(II)(tpy)(GXR)]2+ ions fragment to form odd electron GXR+ radical cations with sufficient abundance to examine their gas-phase fragmentation reactions. The GXR+ radical cations undergo a diverse range of fragmentation reactions which depend on the nature of the side chain of X. Many of these reactions can be rationalized as arising from the intermediacy of isomeric distonic ions in which the charge (i.e. proton) is sequestered by the highly basic arginine side chain and the radical site is located at various positions on the tripeptide including the peptide back bone and side chains. The radical sites in these distonic ions often direct the fragmentation reactions via the expulsion of small radicals (to yield even electron ions) or small neutrals (to form radical cations). Both classes of reaction can yield useful structural information, allowing for example, distinction between leucine and isoleucine residues. The gas-phase fragmentation reactions of the GXR+ radical cations are also compared to their even electron [GXR+H]+ and [GXR+2H]2+ counterparts. The [GXR+H]+ ions give fewer sequence ions and more small molecule losses while the [GXR+2H]2+ ions yield more sequence information, consistent with the ‘mobile proton model’ described in previous studies. In general, all three classes of ions give complementary structural information, but the GXR+ radical cations exhibit a more diverse loss of small species (radicals and neutrals). Finally, links between these gas-phase results and key radical species derived from amino acids, peptides and proteins described in the literature are made.
A modified Finnigan LCQ quadrupole ion trap has been used to determine the equilibrium constant of the complexation reaction of thiophenolate with 2,2,2-trifluoroethanol. The process is particularly useful as a thermometer reaction because it has an exceptionally large temperature dependence. Using literature values for the thermochemistry, an effective ion temperature of 310 ± 20 K is indicated for the ion trap. This value is much lower than some earlier estimates for ion traps, but is consistent with a recent theoretical analysis and some previous interpretations of experimental data. The results suggest that quadrupole ion traps are suitable for studying gas phase reactions under nearly thermal conditions.
The MD+ ions of a variety of amino acids and small peptides have been prepared using CD4 and (CD3)2CO as chemical ionization reagents. Using tandem mass spectrometry the fragmentation reactions of these MD+ ions have been studied, both those occurring unimolecularly on the metastable ion time scale (CD4 CI) and those occurring following collisional activation ((CD3)2CO CI). The results show that the added D+ has undergone extensive interchange (leading to H/D scrambling) with all labile hydrogens including carboxylic hydrogens, hydroxylic hydrogens, amidic hydrogens and amino hydrogens. The results indicate that the proton added to amino acids and simple peptides is very mobile and samples all positions bearing labile hydrogens prior to fragmentation of the protonated species.
An unprecedented method of producing molecular radical cations of oligopeptides in the gas phase has been discovered. Electrospraying a methanolic mixture of a Cu(II)-amine complex, e.g., Cu-II(dien)(NO3)(2) (where dien = diethylenetriamine), and an oligopeptide (M) yields the [Cu-II(dien)M](. 2+) ion, whose collision-induced dissociation (CID) produces [Cu-I(dien)](+) and M.+, the molecular cation of the oligopeptide. Abundant M.+ is apparent when the oligopeptide contains both a tyrosyl and a basic residue-arginyl, lysyl, or histidyl. These structural requirements are similar to those in the metalloradical enzyme process in photosystem II. Tandem mass spectrometry of M.+ produces fragment ions that are both common to and also different from [M + H](+). The fragmentation chemistry of M.+ and of its products appear to be radical driven.
A crown ether based, photolabile radical precursor which forms noncovalent complexes with peptides has been prepared. The peptide/precursor complexes can be electrosprayed, isolated in an ion trap, and then subjected to laser photolysis and collision induced dissociation to generate hydrogen deficient peptide radicals. It is demonstrated that these peptide radicals behave very differently from the hydrogen rich peptide radicals generated by electron capture methods. In fact, it is shown that side chain chemistry dictates both the occurrence and relative abundance of backbone fragments that are observed. Fragmentation at aromatic residues occurs preferentially over most other amino acids. The origin of this selectivity relates to the mechanism by which backbone dissociation is initiated. The first step is abstraction of a beta-hydrogen from the side chain, followed by beta-elimination to yield primarily a-type fragment ions. Calculations reveal that those side chains which can easily lose a beta-hydrogen correlate well with experimentally favored sites for backbone fragmentation. In addition, radical mediated side chain losses from the parent peptide are frequently observed. Eleven amino acids exhibit unique mass losses from side chains which positively identify that particular amino acid as part of the parent peptide. Therefore, side chain losses allow one to unambiguously narrow the possible sequences for a parent peptide, which when combined with predictable backbone fragmentation should lead to greatly increased confidence in peptide identification.
The factors that control the reactivities of aryl radicals toward hydrogen-atom donors were studied by using a dual-cell Fourier-transform ion cyclotron resonance mass spectrometer. Hydrogen-atom abstraction reaction efficiencies for two substrates, cyclohexane and isopropyl alcohol, were measured for 23 structurally different, positively charged aryl radicals, which included dehydrobenzenes, dehydronaphthalenes, dehydropyridines, and dehydro(iso)quinolines. A logarithmic correlation was found between the hydrogen-atom abstraction reaction efficiencies and the (calculated) vertical electron affinities (EA) of the aryl radicals. Transition state energies calculated for the reaction of three of the aryl radicals with isopropyl alcohol were found to correlate linearly with their (calculated) EAs. No correlation was found between the hydrogen-atom abstraction reaction efficiencies and the (calculated) enthalpy changes for the reactions. Measurement of the reaction efficiencies for the reactions of 15 different hydrogen-atom donors with two selected aryl radicals revealed a logarithmic correlation between the hydrogen-atom abstraction reaction efficiencies and the vertical ionization energies (IE) of the hydrogen-atom donors, but not the lowest homolytic X-H (X = heavy atom) bond dissociation energies of the hydrogen-atom donors. Examination of the hydrogen-atom abstraction reactions of 29 different aryl radicals and 18 different hydrogen-atom donors showed that the reaction efficiency increases (logarithmically) as the difference between the IE of the hydrogen-atom donor and the EA of the aryl radical decreases. This dependence is likely to result from the increasing polarization, and concomitant stabilization, of the transition state. Thus, the hydrogen-atom abstraction reaction efficiency for an aryl radical can be "tuned" by structural changes that influence either the vertical EA of the aryl radical or the vertical IE of the hydrogen atom donor.
Collisional electron transfer from gaseous Cs atoms was studied for singly and doubly protonated peptides Gly-Arg (GR) and Ala-Arg (AR) at 50- and 100-keV kinetic energies. Singly protonated GR and AR were discharged to radicals that in part rearranged by migration of a C(alpha) hydrogen atom onto the guanidine group. The C(alpha)-radical isomers formed were detected as stable anions following transfer of a second electron. In addition to the stabilizing rearrangements, the radicals underwent side-chain and backbone dissociations. The latter formed z fragments that were detected as the corresponding anions. Analysis of the (GR + H)(.) radical potential energy surface using electronic structure theory in combination with Rice-Ramsperger-Kassel-Marcus calculations of rate constants indicated that the arginine C(alpha) hydrogen atom was likely to be transferred to the arginine side-chain on the experimental timescale of <or=200 ns. Transfer of the Gly C(alpha)H was calculated to have a higher transition-state energy and was not kinetically competitive. Collisional electron transfer to doubly protonated GR and AR resulted in complete dissociation of (GR + 2H)(+.) and (AR + 2H)(+.) ions by loss of H, ammonia, and NC(alpha) bond cleavage. Electronic structure theory analysis of (GR + 2H)(+.) indicated the presence of multiple conformers and electronic states that differed in reactivity and steered the dissociations to distinct channels. Electron attachment to (GR + 2H)(2+) resulted in the formation of closely spaced electronic states of (GR + 2H)(+.) in which the electron density was delocalized over the guanidinium, ammonium, amide, and carboxyl groups. The different behavior of (GR + H)(.) and (GR + 2H)(+.) is explained by the different timescales for dissociation and different internal energies acquired upon electron transfer.
The effects of water on electron capture dissociation products, molecular survival, and recombination energy are investigated for diprotonated Lys-Tyr-Lys solvated by between zero and 25 water molecules. For peptide ions with between 12 and 25 water molecules attached, electron capture results in a narrow distribution of product ions corresponding to primarily the loss of 10-12 water molecules from the reduced precursor. From these data, the recombination energy (RE) is determined to be equal to the energy that is lost by evaporating on average 10.7 water molecules, or 4.3 eV. Because water stabilizes ions, this value is a lower limit to the RE of the unsolvated ion, but it indicates that the majority of the available RE is deposited into internal modes of the peptide ion. Plotting the fragment ion abundances for ions formed from precursors with fewer than 11 water molecules as a function of hydration extent results in an energy resolved breakdown curve from which the appearance energies of the b 2 (+), y 2 (+), z 2 (+*), c 2 (+), and (KYK + H) (+) fragment ions formed from this peptide ion can be obtained; these values are 78, 88, 42, 11, and 9 kcal/mol, respectively. The propensity for H atom loss and ammonia loss from the precursor changes dramatically with the extent of hydration, and this change in reactivity can be directly attributed to a "caging" effect by the water molecules. These are the first experimental measurements of the RE and appearance energies of fragment ions due to electron capture dissociation of a multiply charged peptide. This novel ion nanocalorimetry technique can be applied more generally to other exothermic reactions that are not readily accessible to investigation by more conventional thermochemical methods.
DFT calculations have been performed with the B3LYP and MPW1K functional on the hydrogen atom abstraction reactions of ethenoxyl with ethenol and of phenoxyl with both phenol and alpha-naphthol. Comparison with the results of G3 calculations shows that B3LYP seriously underestimates the barrier heights for the reaction of ethenoxyl with ethenol by both proton-coupled electron transfer (PCET) and hydrogen atom transfer (HAT) mechanisms. The MPW1K functional also underestimates the barrier heights, but by much less than B3LYP. Similarly, comparison with the results of experiments on the reaction of phenoxyl radical with alpha-naphthol indicates that the barrier height for the preferred PCET mechanism is calculated more accurately by MPW1K than by B3LYP. These findings indicate that the MPW1K functional is much better suited than B3LYP for calculations on hydrogen abstraction reactions by both HAT and PCET mechanisms.
Site-specific fragmentation of peptides at phosphorylated serine or threonine residues is demonstrated. This radical directed cleavage is accomplished by a two-step procedure. First the phosphate is replaced with naphthalenethiol using well established Michael Addition chemistry. Second, the modified peptide is electrosprayed and subjected to irradiation at 266 nm. Absorption at naphthalene causes homolytic cleavage of the connecting carbon-sulfur bond yielding a radical in the beta-position. Subsequent rearrangement cleaves the peptide backbone yielding a d-type fragment. This chemistry is generally applicable as demonstrated by experiments with several different peptides. Assignment of phosphorylation sites is greatly facilitated by this approach, particularly for peptides containing multiple serine or threonine residues.
The mechanism for the formation of y ions in the collision-induced dissociation (CID) spectra of protonated peptides produced by fast-atom bombardment was investigated by tandem mass spectrometry and deuterium labelling studies. The results show that a hydrogen atom attached to nitrogen and not to carbon migrates during cleavage of the amide bond. A mechanism based on these results is presented.
The bond dissociation enthalpies (BDE) of all of the amino acid residues, modeled by HC(O)NHCH(R)C(O)NH(2) (PH(res)), were determined at the B3LYP/6-31G//B3LYP/6-31G level, coupled with isodesmic reactions. The results for neutral side chains with phi, psi angles approximately 180 degrees, approximately 180 degrees in ascending order, to an expected accuracy of +/-10 kJ mol(-)(1), are Asn 326; cystine 330; Asp 332; Gln 334; Trp 337; Arg 340; Lys 340; Met 343; His 344; Phe 344; Tyr 344; Leu 344; Ala 345; Cys 346; Ser 349; Gly 350; Ile 351; Val 352; Glu 354; Thr 357; Pro-cis 358; Pro-trans 369. BDEs calculated at the ROMP2/6-31G//B3LYP/6-31G level exhibit the same trends but are approximately 7 kJ mol(-)(1) higher. All BDEs are smaller than those of typical secondary or tertiary C-H bonds due to the phenomenon of captodative stabilization. The stabilization is reduced by changes in the phi,psi angles. As a result the BDEs increase by about 10 kJ mol(-)(1) in beta-sheet and 40 kJ mol(-)(1) in alpha-helical environments, respectively. In effect the alpha C-H BDEs can be "tuned" from about 345 to 400 kJ mol(-)(1) by adjusting the local environment. Some very significant effects of this are seen in the current literature on H-transfer processes in enzyme mechanisms and in oxidative damage to proteins. These observations are discussed in terms of the findings of the present study.
In this Account we have compiled a list of reliable bond energies that are based on a set of critically evaluated experiments. A brief description of the three most important experimental techniques for measuring bond energies is provided. We demonstrate how these experimental data can be applied to yield the heats of formation of organic radicals and the bond enthalpies of more than 100 representative organic molecules.
For small cyclic peptides, one electron capture by the [M + 2H](2+) ion generates numerous fragments corresponding to amino acid losses, side-chain losses, and losses of some low molecular weight species such as H(2)O, CH(3)(*), C(3)H(6), and (*)CONH(2). As predicted, the side-chain cleavages are amplified relative to linear peptides of similar size, but the amino acid losses were unexpected because they require that one electron capture cause more than one backbone cleavage, a phenomenon which necessitates further refinement or reinterpretation of current ECD mechanisms. A modified mechanism is postulated in which nonergodic electron capture fragmentation generates an alpha-carbon radical species that then propagates along the protein backbone. This radical migration initiates multiple free radical rearrangements, which cause both multiple backbone cleavages and additional side-chain cleavages.
The mechanism of the cleavage of protonated amide bonds of oligopeptides is discussed in detail exploring the major energetic, kinetic, and entropy factors that determine the accessibility of the b(x)-y(z) (Paizs, B.; Suhai, S. Rapid Commun. Mass Spectrom. 2002, 16, 375) and "diketopiperazine" (Cordero, M. M.; Houser, J. J.; Wesdemiotis, C. Anal. Chem. 1993, 65, 1594) pathways. General considerations indicate that under low-energy collision conditions the majority of the sequence ions of protonated oligopeptides are formed on the b(x)-y(z) pathways which are energetically, kinetically, and entropically accessible. This is due to the facts that (1).the corresponding reactive configurations (amide N protonated species) can easily be formed during ion excitation, (2). most of the protonated nitrogens are stabilized by nearby amide oxygens making the spatial arrangement of the two amide bonds (the protonated and its N-terminal neighbor) involved in oxazolone formation entropically favored. On the other hand, formation of y ions on the diketopiperazine pathways is either kinetically or energetically or entropically controlled. The energetic control is due to the significant ring strain of small cyclic peptides that are co-formed with y ions (truncated protonated peptides) similar in size to the original peptide. The entropy control precludes formation of y ions much smaller than the original peptide since the attacking N-terminal amino group can rarely get close to the protonated amide bond buried by amide oxygens. Modeling the b(x)-y(z) pathways of protonated pentaalanine leads for the first time to semi-quantitative understanding of the tandem mass spectra of a protonated oligopeptide. Both the amide nitrogen protonated structures (reactive configurations for the amide bond cleavage) and the corresponding b(x)-y(z) transition structures are energetically more favored if protonation occurs closer to the C-terminus, e.g., considering these points the Ala(4)-Ala(5) amide bond is more favored than Ala(3)-Ala(4), and Ala(3)-Ala(4) is more favored than Ala(2)-Ala(3). This fact explains the increasing ion abundances observed for the b(2)/y(3), b(3)/y(2), and b(4)/y(1) ion pairs in the metastable ion and low-energy collision induced mass spectra (Yalcin, T.; Csizmadia, I. G.; Peterson, M. B.; Harrison, A. G. J. Am. Soc. Mass Spectrom. 1996, 7, 233) of protonated pentaalanine. A linear free-energy relationship is used to approximate the ratio of the b(x) and y(z) ions on the particular b(x)-y(z) pathways. Applying the necessary proton affinities such considerations satisfactorily explain for example dominance of the b(4) ion over y(1) and the similar b(3) and y(2) ion intensities observed for the metastable ion and low-energy collision induced mass spectra.
Strategies are reported that combine in one step a predictable chemical-based protein digestion with mass spectrometry. Lysine residue amino groups in peptides and proteins are modified by reaction with a peroxycarbonate derived from p-nitrophenol, and tert-butyl hydroperoxide. The peroxycarbonate reacts with lysine residues in peptides and proteins, and the resulting lysine peroxycarbamates undergo homolytic fragmentation under conditions of low-energy collision-induced dissociation (CID). Observed fragmentation of the peptides involves apparent free radical processes including Hofmann-Löffler-type rearrangements that lead to peptide chain fragmentation. Strategies for directed cleavage of peptides by free radical promoted processes are feasible, and such strategies may well simplify schemes for protein analysis.
A study in contrasts: Mass spectra of singly protonated peptide ions excited with 157-nm light show unorthodox fragmentation patterns in comparison to those obtained by collision-induced dissociation (CID). Observations are consistent with an initial photochemical cleavage of α-C-CO bonds in the peptide backbone by a Norrish Type I reaction to create radical precursor ions followed by one of three elimination processes that form ion fragments with even numbers of electrons.
The effects of positive charge on the properties of ammonium and amide radicals were investigated by ab initio and density functional theory calculations with the goal of elucidating the energetics of electron capture dissociation (ECD) of multiply charged peptide ions. The electronic properties of the amide group in N-methylacetamide (NMA) are greatly affected by the presence of a remote charge in the form of a point charge, methylammonium, or guanidinium cations. The common effect of the remote charge is an increase of the electron affinity of the amide group, resulting in exothermic electron capture. The N—Cα
bond dissociation and transition state energies in charge-stabilized NMA anions are 20–50 kJ mol−1 greater than in the hydrogen atom adduct. The zwitterions formed by electron capture have proton affinities that were calculated as 1030–1350 kJ mol−1, and are sufficiently basic for the amide carbonyl to exothermically abstract a proton from the ammonium, guanidinium and imidazolium groups in protonated lysine, arginine, and histidine residues, respectively. A new mechanism is proposed for ECD of multiply charged peptide and protein cations in which the electron enters a charge-stabilized electronic state delocalized over the amide group, which is a superbase that abstracts a proton from a sterically proximate amino acid residue to form a labile aminoketyl radical that dissociates by N—Cα
bond cleavage. This mechanism explains the low selectivity of N—Cα
bond dissociations induced by electron capture, and is applicable to dissociations of peptide ions in which the charge carriers are metal ions or quaternary ammonium groups. The new amide superbase and the previously proposed mechanisms of ECD can be uniformly viewed as being triggered by intramolecular proton transfer in charge-reduced amide cation-radicals. In contrast, remote charge affects N—H bond dissociation in weakly bound ground electronic states of hypervalent ammonium radicals, as represented by methylammonium, CH3NH
3·, but has a negligible effect on the N—H bond dissociation in the strongly bound excited electronic states. This refutes previous speculations that loss of “hot hydrogen” can occur from an excited state of an ammonium radical.
The photodissociation by 157 nm light of singly- and doubly-charged peptide ions containing C- or N-terminal arginine residues was studied in a linear ion trap mass spectrometer. Singly-charged peptides yielded primarily x- and a-type ions, depending on the location of the arginine residue, along with some related side-chain fragments. These results are consistent with our previous work using a tandem time-of-flight (TOF) instrument with a vacuum matrix-assisted laser desorption/ionization (MALDI) source. Thus, the different internal energies of precursor ions in the two experiments seem to have little effect on their photofragmentation. For doubly-charged peptides, the dominant fragments observed in both photodissociation and collisionally induced dissociation (CID) experiments are b- and y-type ions. Preliminary experiments demonstrating fragmentation of multiply-charged ubiquitin ions by 157 nm photodissociation are also presented.
Loss of side chains from different amino acid residues in a model peptide framework of RGGGXGGGR under electron capture dissociation conditions were systematically investigated, where X represents one of the twenty common amino acid residues. The alpha-carbon radical cations initially formed by N-Calpha cleavage of peptide ions were shown to undergo secondary dissociation through losses of even-electron and/or odd-electron side-chain moieties. Among the twenty common amino acid residues studied, thirteen of them were found to lose their characteristic side chains in terms of odd-electron neutral fragments, and nine of them were found to lose even-electron neutral side chains. Several generalized dissociation pathways were proposed and were evaluated theoretically with truncated leucine-containing models using ab initio calculations at B3-PMP2/6-311++G(3df,2p)//B3LYP/6-31++G(d,p) level. Elimination of odd-electron side chain was associated with the initial abstraction of the hydrogen from the alpha-carbon bearing the side chain by the N-terminal alpha-carbon radical. Subsequent formation of alpha-beta carbon-carbon double bond leads to the elimination of the odd-electron side chain. The energy barrier for this reaction pathway was 89 kJmol-1. This reaction pathway was 111 kJmol-1 more favorable than the previously proposed pathway involving the formation of cyclic lactam. Elimination of even-electron side chain was associated with the initial abstraction of the gamma-hydrogen from the side chain by the N-terminal alpha-carbon radical. Subsequent formation of beta-gamma carbon-carbon double bond leads to the elimination of the even-electron side chain and the migration of the radical center to the alpha-carbon. The energy barrier for this fragmentation reaction was found to be 50 kJmol-1.
The free radical initiator Vazo 68 is coupled to a peptide and electrosprayed into an ion trap mass spectrometer. On collisional activation, the Vazo 68-peptide conjugate generates a free radical, which can be collisionally activated to cleave the peptide backbone. Mostly z-type fragments are formed, as in CAD of other radical peptides and ECD fragmentation. We present data for the Angiotensin II-Vazo 68 conjugate and discuss possible sites of H atom abstraction from the peptide. This experimental methodology for generating peptide fragments is a useful step toward the development of a completely gas-phase approach to protein sequencing.
To explore the mechanism of electron capture dissociation (ECD) of linear peptides, a set of 16-mer peptides were synthesized with deuterium labeled on the alpha-carbon position of four glycines. The ECD spectra of these peptides showed that such peptides exhibit a preference for the radical to migrate to the alpha-carbon position on glycine via hydrogen (or deuterium) abstraction before the final cleavage and generation of the detected product ions. The data show c-type fragment ions, ions corresponding to the radical cation of the c-type fragments, c*, and they also show c*-1 peaks in the deuterated peptides only. The presence of the c*-1 peaks is best explained by radical-mediated scrambling of the deuterium atoms in the long-lived, metastable, radical intermediate complex formed by initial electron capture, followed by dissociation of the complex. These data suggest the presence of at least two mechanisms, one slow, one fast. The abundance of H* and -CO losses from the precursor ion changed upon deuterium labeling indicating the presence of a kinetic isotope effect, which suggests that the values reported here represent an underestimation of radical migration and H/D scrambling in the observed fragments.
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.
High-level quantum chemistry calculations have been carried out to investigate beta-scission reactions of alkoxyl radicals located at the alpha-carbon of a peptide backbone. This type of alkoxyl radical may undergo three possible beta-scission reactions, namely C-C beta-scission of the backbone, C-N beta-scission of the backbone, and C-R beta-scission of the side chain. We find that the rates for the C-C beta-scission reactions are all very fast, with rate constants of the order 10(12) s(-1) that are essentially independent of the side chain. The C-N beta-scission reactions are all slow, with rate constants that range from 10(-0.7) to 10(-4.5) s(-1). The rates of the C-R beta-scission reactions depend on the side chain and range from moderately fast (10(7) s(-1)) to very fast (10(12) s(-1)). The rates of the C-R beta-scission reactions correlate well with the relative stabilities of the resultant side-chain product radicals (*R), as reflected in calculated radical stabilization energies (RSEs). The order of stabilities for the side-chain fragment radicals for the natural amino acids is found to be Ala < Glu < Gln approximately Leu approximately Met approximately Lys approximately Arg < Asp approximately Ile approximately Asn approximately Val < Ser approximately Thr approximately Cys < Phe approximately Tyr approximately His approximately Trp. We predict that for side-chain C-R beta-scission reactions to effectively compete with the backbone C-C beta-scission reactions, the side-chain fragment radicals would generally need an RSE greater than approximately 30 kJ mol(-1). Thus, the residues that may lead to competitive side-chain beta-scission reactions are Ser, Thr, Cys, Phe, Tyr, His, and Trp.
The combined use of advanced mass spectrometry experiments, condensed-phase synthesis of serine and homoserine nitrate ester radical precursors, and high-level ab initio calculations provides a powerful way of examining the fundamental reactivity of radicals derived from peptides.
Electron capture dissociation was studied with tetradecapeptides and pentadecapeptides that were capped at N-termini with a 2-(4′-carboxypyrid-2′-yl)-4-carboxamide group (pepy), e. g., pepy-AEQLLQEEQLLQEL-NH2, pepy-AQEFGEQGQKALKQL-NH2, and pepy-AQEGSEQAQKFFKQL-NH2. Doubly and triply protonated peptide cations underwent efficient electron capture in the ion-cyclotron resonance cell to yield charge-reduced species. However, the electron capture was not accompanied by backbone dissociations. When the peptide ions were preheated by absorption of infrared photons close to the dissociation threshold, subsequent electron capture triggered ion dissociations near the remote C-terminus forming mainly (b
11–14+1)+· fragment ions that were analogous to those produced by infrared multiphoton dissociation alone. Ab initio calculations indicated that the N-1 and N-1′ positions in the pepy moiety had topical gas-phase basicities (GB=923 kJ mol−1) that were greater than those of backbone amide groups. Hence, pepy was a likely protonation site in the doubly and triply charged ions. Electron capture in the protonated pepy moiety produced the ground electronic state of the charge-reduced cation-radical with a topical recombination energy, RE=5.43–5.46 eV, which was greater than that of protonated peptide residues. The hydrogen atom in the charge-reduced pepy moiety was bound by >160 kJ mol−1, which exceeded the hydrogen atom affinity of the backbone amide groups (21–41 kJ mol−1). Thus, the pepy moiety functioned as a stable electron and hydrogen atom trap that did not trigger radical-type dissociations in the peptide backbone that are typical of ECD. Instead, the internal energy gained by electron capture was redistributed over the peptide moiety, and when combined with additional IR excitation, induced proton-driven ion dissociations which occurred at sites that were remote from the site of electron capture. This example of a spin-remote fragmentation provided the first clear-cut experimental example of an ergodic dissociation upon ECD.
Achieving a fundamental understanding of the mechanism of unimolecular dissociation of internally excited complex molecules is one of the most important challenges in modern mass spectrometry. One of the central questions is whether the dissociation of large molecules is properly described by statistical theoriesâRRKM/QET or Phase Space Theories âthat have proved to be remarkably successful both for small molecules and a number of small and medium size peptides. The concept question is whether the ergodic assumption that the internal excitation of the ion is randomly redistributed among the vibrational degrees of freedom prior to fragmentation is satisfied for large molecules. The validity of the ergodic hypothesis for dissociation of gas-phase biomolecules has been recently reviewed and will be only briefly discussed here.
Comparison between the gas-phase fragmentation of odd-electron M+*, [M + H]2+*, and [M - 2H]-* ions of model peptides suggests that charge-remote radical-driven fragmentation pathways play an important role in the dissociation of odd-electron peptide ions. We have found that charge-remote processes are responsible for a variety of side-chain losses from the precursor ion and some backbone fragmentation. These fragmentation pathways most likely involve hydrogen abstraction by the radical site that initiates subsequent cleavages. These findings are generally relevant to our understanding of the fragmentation patterns of odd-electron peptide ions produced through various approaches including the capture of low-energy electrons, electron detachment, and electron transfer.
Time- and collision energy-resolved surface-induced dissociation (SID) of ternary complexes of Co(III)(salen)+, Fe(III)(salen)+, and Mn(III)(salen)+ with several angiotensin peptide analogues was studied using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. Time-resolved fragmentation efficiency curves (TFECs) were modeled using an RRKM-based approach developed in our laboratory. The approach utilizes a very flexible analytical expression for the internal energy deposition function that is capable of reproducing both single-collision and multiple-collision activation in the gas phase and excitation by collisions with a surface. The energetics and dynamics of competing dissociation pathways obtained from the modeling provides important insight on the competition between proton transfer, electron transfer, loss of neutral peptide ligand, and other processes that determine gas-phase fragmentation of these model systems. Similar fragmentation behavior was obtained for various Co(III)(salen)-peptide systems of different angiotensin analogues. In contrast, dissociation pathways and relative stabilities of the complexes changed dramatically when cobalt was replaced with trivalent iron or manganese. We demonstrate that the electron-transfer efficiency is correlated with redox properties of the metal(III)(salen) complexes (Co > Fe > Mn), while differences in the types of fragments formed from the complexes reflect differences in the modes of binding between the metal-salen complex and the peptide ligand. RRKM modeling of time- and collision-energy-resolved SID data suggests that the competition between proton transfer and electron transfer during dissociation of Co(III)(salen)-peptide complexes is mainly determined by differences in entropy effects while the energetics of these two pathways are very similar.
The 9-fluorenylmethyloxycarbonyl aminoprotecting group
- L A Carpino
- G Y Han
Carpino, L. A.; Han, G. Y. The 9-fluorenylmethyloxycarbonyl aminoprotecting group. J. Org. Chem. 1972, 37, 3404 -3408.