Advertisement

Journal of The American Society for Mass Spectrometry

, Volume 27, Issue 9, pp 1454–1467 | Cite as

Ground and Excited-Electronic-State Dissociations of Hydrogen-Rich and Hydrogen-Deficient Tyrosine Peptide Cation Radicals

  • Emilie Viglino
  • Cheuk Kuen Lai
  • Xiaoyan Mu
  • Ivan K. Chu
  • František Tureček
Research Article

Abstract

We report a comprehensive study of collision-induced dissociation (CID) and near-UV photodissociation (UVPD) of a series of tyrosine-containing peptide cation radicals of the hydrogen-rich and hydrogen-deficient types. Stable, long-lived, hydrogen-rich peptide cation radicals, such as [AAAYR + 2H]+● and several of its sequence and homology variants, were generated by electron transfer dissociation (ETD) of peptide-crown-ether complexes, and their CID-MS3 dissociations were found to be dramatically different from those upon ETD of the respective peptide dications. All of the hydrogen-rich peptide cation radicals contained major (77%–94%) fractions of species having radical chromophores created by ETD that underwent photodissociation at 355 nm. Analysis of the CID and UVPD spectra pointed to arginine guanidinium radicals as the major components of the hydrogen-rich peptide cation radical population. Hydrogen-deficient peptide cation radicals were generated by intramolecular electron transfer in CuII(2,2:6,2-terpyridine) complexes and shown to contain chromophores absorbing at 355 nm and undergoing photodissociation. The CID and UVPD spectra showed major differences in fragmentation for [AAAYR]+● that diminished as the Tyr residue was moved along the peptide chain. UVPD was found to be superior to CID in localizing Cα-radical positions in peptide cation radical intermediates.

Graphical Abstract

Keywords

Tyrosine peptides Peptide cation radicals Photodissociation 

Introduction

Whereas isolated peptide molecules and ions are inherently stable species with closed-electronic shells, perturbation of their electronic structure results in novel and often unexpected chemistry [1]. There are now several ways of generating peptide radicals and cation radicals in the gas phase to be investigated by mass spectrometry. Resonant multiphoton ionization of gas-phase peptide molecules is the most direct method that was reported as early as in 1986 [2, 3, 4]. One-electron oxidation of peptide ligands upon collision-induced dissociation of ternary complexes of transition metal ions represents another useful method for generating peptide cation radicals [5, 6, 7, 8, 9]. Thermal [10] or light-induced [11] homolytic bond dissociations in suitably derivatized peptide ions have also been developed into methods of peptide cation radical generation. All these techniques produce peptide cation radicals that are stoichiometrically equivalent to neutral peptide molecules and because they do not contain additional hydrogens from protonation, they are called “hydrogen deficient” [1]. Of note is that the structures of hydrogen-deficient peptide cation radicals, wherever the charge and radical sites are known, depend on the particular activation method.

A fundamentally different method of generating peptide cation radicals relies on electron attachment to multiply protonated peptides via capture of a slow free electron [12] or collisional transfer from a suitable neutral [13, 14, 15, 16, 17, 18] or anionic donor [19]. Peptide cation radicals of this type are called “hydrogen rich.” They are often unstable and undergo extensive dissociations by multiple channels forming backbone fragment ions and providing sequence information for protein analysis [1]. The majority of cation radical fragment ions produced by electron attachment arise by cleavage of bonds between an amide nitrogen and the Cα atom of the adjacent amino acid residue, N–Cα cleavage for short, retaining the radical site in C-terminal residues (z-type ions) [20]. When singly protonated z ions are stoichiometrically equivalent to deaminated hydrogen-deficient peptide cation radicals, they have been considered as representing a connecting bridge between the chemistries of both types of peptide cation radicals [21].

Selected peptide cation radicals of either type have been studied with the aim of determining their structure and elucidating dissociation mechanisms [22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32]. However, despite there being a large body of data on peptide cation radicals and substantial progress in understanding their chemistry, there has not been a study exploring side by side the properties of both hydrogen-deficient and hydrogen-rich peptide cation radicals derived from the same peptide molecules. Here, we report a comparative study of the generation and unimolecular dissociations of a series of tyrosine containing peptides in their hydrogen-rich and hydrogen-deficient radical forms. We adopt the standard approach of systematically varying the amino acid sequence in Ala-Ala-Ala-Ala-Tyr-Arg (AAAYR) to generate and study both types of derived cation radicals, as well as those from sequence isomers AAYAR, AYAAR, and YAAAR. The C-terminal arginine residue is employed throughout to anchor the charging proton at a specific site. Substitution of Val for Ala is used in AVAYR, AAYVR, AYAVR, and YAAVR to aid fragment ion identification through predictable mass shifts. Specific methods of generating these peptide cation radicals are reported, and their dissociations are studied in ground electronic states using collisional activation and in excited electronic states using UV-VIS photodissociation at wavelengths targeting the radical chromophores.

Experimental

Materials and Methods

All peptides were synthesized on Wang resin (Bachem Americas, Torrance, CA, USA) using commercially available Fmoc peptides (Life Technologies, Rockford, IL, USA) and purified by ion-exchange chromatography. Fmoc-(O-methyl)-L-tyrosine was purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA) and used in standard solid-state peptide synthesis of AAY(OCH3)R. 2,2′:6′,2″-Terpyridine and Cu(NO3)2 were obtained from Sigma-Aldrich (St. Louis, MO, USA). When generating peptide radicals using metal complexes, 600 μM CuII(tpy)(NO3)2 was added to the peptide stock solutions, without acetic acid, such that peptide radical ions (M+●) would be generated through one-electron transfer from the neutral peptide to the metal center in the metal–tpy–peptide ternary complex. For the experiments performed with the triple-quadrupole instrument, [CuII(tpy)M]2+● was first introduced to the ion source by the electrospray with an ion spray voltage of 3.5 kV. The M+● species was then generated through in-source fragmentations of the complexes using N2 as the collision gas under a declustering potential of 35 eV. The resulting M+● species was selected in the first quadrupole (Q1) and further dissociated in the collision cell under the MS2 mode [33]. The specific α-carbon-centered radicals were formed through multistage CID of [CuII(tpy)(M)]2+●, in which peptide canonical radical cations M+● were generated in the first stage of CID of metal–tpy–peptide complexes and then underwent a subsequent stage of CID to generate the α-carbon-centered radical through tyrosine side-chain loss. For the experiments performed with the LTQ-XL linear ion trap mass spectrometer, electrospray ionization with a home-built microspray source was used to generate gas-phase [CuII(tpy)peptide]2+● ions according to the above-described procedure. The 65Cu or 63Cu isotopologues were selected by mass and subjected to collision-induced dissociation (CID) at collision energies that were tuned to optimize peptide cation-radical formation, typically at normalized collision energies (NCE) set to 15–18 instrument units. The [peptide]+● ions at the corresponding m/z were selected by mass and subjected to CID or photodissociation (UVPD). Electron transfer dissociation mass spectra were obtained and carried out on a modified LTQ-XL linear ion trap mass (LIT) spectrometer (ThermoElectron Fisher, San Jose, CA, USA) equipped with a laser system. Doubly charged peptide ions or their crown-ether complexes were produced by electrospray ionization, mass-selected in the ion trap and allowed to react with fluoranthene anions at typical reaction times of 200 ms. The CID and UVPD mass spectra of mass-selected peptide cation radicals were measured on the LTQ-XL. High-resolution mass spectra were measured on an LTQ-Orbitrap (ThermoElectron Fisher, San Jose, CA, USA). The peptide cation-radicals were prepared by CID in the LTQ and transferred to the Orbitrap for high-resolution measurements using Fourier-transform treatment of the time-domain signal. The mass resolution was set to 100,000.

Photodissociation

Photodissociation of trapped ions in the LIT was performed as reported previously [34]. The typical experimental setup consists of selecting the ion to be photodissociated and storing it in the LIT for a chosen time period. For example, 400-ms storage time can accommodate up to seven laser pulses spaced by 50 ms. This allows one to vary the number of pulses, which are also normalized as the number of pulses used is varied depending on the degree of dissociation. The pulse-dependent UVPD measurements were performed with the 355 nm line from the laser source at 15 mJ/pulse laser power, as described previously [34].

Calculations

Standard ab initio and density functional theory (DFT) calculations were carried out using the Gaussian 09 suite of programs [35]. Ion structures were gradient-optimized with the B3LYP [36], M06-2X [37], and ωB97X-D [38] hybrid DFT methods all using the 6-31 + G(d,p) basis set, and local energy minima were confirmed by harmonic frequency analysis. Single-point energy calculations were conducted with DFT and Møller-Plesset perturbational treatment [39] (MP2, frozen core) using the 6-311++G(2d,p) basis set.

Note on Nomenclature

To describe in a uniform way the fragment ions originating from different types of peptide cation-radicals, we adopt the all-inclusive nomenclature system introduced recently [40]. Briefly, this uses the original Biemann system of naming peptide fragments according to the backbone bond being broken [20], but explicitly assigning the charge, radical, and number of hydrogen atoms included in the fragment ion [40]. The equivalent names for singly charged fragment ions are as follows (n = number of residues): [a n ]+ = a n, [b n ]+ = b n, [c n + 2H]+ = c n, [c n + H]+● = c n − 1, [x n + H]+● = x n, [y n + 2H]+ = y n, [y n ]+ = y n − 2, [z n + H]+● = z n, [z n + 2H]+ = z n + 1. Residues in fragment ions arising by side-chain loss from Tyr are named Gα .

Results

Hydrogen-Rich Peptide Cation-Radicals

Electron transfer dissociation of the doubly charged tyrosine peptide ions resulted in extensive dissociation, forming abundant [z 1 -z 4 + H]+● sequence fragment ions, but leaving very weak survivor [M + 2H]+● peptide cation radicals that overlapped with residual 13C isotope satellites of the [M + H]+ fragment ions (Figure S1a–d, Supplementary Material). To increase the [M + 2H]+● yield upon ETD, we resorted to a previously developed technique in which doubly charged noncovalent peptide 1:1 complexes with 18-crown-6-ether (CE) were treated by ETD [41, 42]. This resulted in charge-reduction and loss of the CE ligand, forming [M + 2H]+● cation radicals at relative abundances that allowed them to be further investigated by tandem (MSn) experiments. In addition to the ligand loss upon reduction, ETD also produced CE-coordinated backbone fragment ions of the N-terminal (c) and C-terminal (z) type (Supplementary Figure S2a–d). These allowed us to estimate the populations of peptide complexes in which the crown ether was attached to the arginine guanidinium and N-terminal ammonium, which are the charged groups in these ions. The data (Supplementary Table S1) indicated somewhat preferential coordination to the arginine charged group (56%–73%) except in AAYAR and AAYVR where the N-terminal coordination was slightly more prevalent (57% and 52%, respectively). The figures for N-terminal coordination should be regarded as lower limits because the ETD spectra of the complexes showed very abundant [M + 2H − NH3]+● fragment ions that could originate from charge-reduced N-terminal CE-H3N- complexes. More discussion of this aspect will be given later in the text.

Collision-induced dissociation of [M + 2H]+● ions generated by ETD of the CE complexes showed a marked dependence on the amino acid sequence (Fig. 1). In general, the MS3/ETD-CID spectra were substantially different from the ETD spectra of [M + 2H]2+ ions as is obvious from the comparison of the Supplementary Figure S1a–d and Fig. 1a–d spectra. The CID fragmentation pattern of [AAAYR + 2H]+● (m/z 552) showed a dominant loss of a hydrogen atom (m/z 551) (Fig. 1a). The other abundant dissociation channels were loss of water (m/z 534), loss of CH5N3 from the arginine side chain (m/z 493), and the formation of the [z 1 + 2H]+ backbone fragment ion at m/z 160. In contrast, backbone fragment ions of the standard [z n + H]+● type (n = 1-4) were not formed. The minor presence of a fragment ion at m/z 444, which corresponds to a loss of 106 Da from the tyrosine side chain (–C7H6O, neutral fragment) is also seen. This loss is commonly observed for hydrogen-deficient tyrosine-containing peptide cation radicals [5, 6, 7, 8, 9], and the ion is denoted as [AAAGα R + H]+, indicating a conversion of the Tyr residue to a glycine Cα-radical [40].
Figure 1

CID-MS3 mass spectra of hydrogen-rich peptide cation-radicals (m/z 552) generated by ETD of crown-ether complexes: (a) [AAAYR + 2H]+●, (b) [AAYAR + 2H]+●, (c) [AYAAR + 2H]+●, (d) [YAAAR + 2H]+●

As tyrosine was moved away from the C-terminus by one residue, the resulting [AAYAR + 2H]+● cation radical showed a different fragmentation pattern upon CID (Fig. 1b). In addition to the same four major channels observed for [AAAYR + 2H]+●, the [AAYAR + 2H]+● fragmentation displayed a greater complexity in the formation of backbone fragments. The major ones were identified as [b 3 ]+ and [b 4 ]+ ions along with [y 3 + 2H]+ and [y 2 + 2H]+ ions, a prominent [y 1 + 2H]+ ion, as well as [z 2 ]+ and [z 3 ]+ ions, and a [a 4 ]+ fragment ion. The dominating dissociation pathways for this sequence were loss of water (m/z 534), the formation of [z 1 + 2H]+ (m/z 160), loss of an H atom (m/z 551), and loss of CH5N3 from the arginine side chain. Also worth noting is the presence of the fragment ion at m/z 444 that results from the tyrosine side chain loss (–C7H6O), which is significantly more abundant than for [AAAYR + 2H]+●.

The dissociation patterns of [AYAAR + 2H]+● and [YAAAR + 2H]+● lack backbone fragments. CID of [AYAAR + 2H]+● resulted in five distinct reactions, with a dominating loss of an H atom and formation of prominent fragment ions at m/z 444, 493, 160, and 534, which were analogous to those described above (Fig. 1c). The same dissociations were observed for [YAAAR + 2H]+●, which showed a more abundant elimination of C7H6O compared with the above-described sequences (Fig. 1d). This dissociation is usually associated with the presence of a tyrosyl-O radical moiety [9, 43]. However, the CID spectra do not allow us to distinguish if Tyr-O radicals were present to a different extent in the stable [M + 2H]+● cation radicals or if they were produced as reactive intermediates in the course of dissociation. The identity of the peptide cation radicals and the assignments of their fragments following CID were corroborated by mass shifts in the CID spectra of cation radicals derived from AVAYR, AAYVR, AYAVR, and YAAVR. The pertinent spectra are presented in the Supplement (Supplementary Figure S3a–d).

UVPD of hydrogen-rich peptide cation radicals was studied at 355 nm, which is a wavelength selectively targeting excitation of peptide radical chromophores [34]. UVPD of [AAAYR + 2H]+● through [YAAAR + 2H]+● (Fig. 2a–d) gave rise to two major fragment ions, one (m/z 551) being formed by photo-induced loss of a hydrogen atom and the other (m/z 160) corresponding to a backbone [z 1 + 2H]+ ion (see inset structure in Fig. 2a). Again, no standard backbone fragment ions of the [z + 2H]+● type were formed, indicating that even the excited electronic states of the long-lived cation-radicals were different from the electronic states accessed by electron transfer to the doubly charged ions in the ETD mode. The UVPD fragment ions were photostable at 355 nm and their relative intensity reached a steady state after four laser pulses (Supplementary Figure S4a–d). Photodissociation of the peptide cation radicals at 355 nm must be associated with a chromophore formed by electron attachment, as the natural amino acid residues Ala, Tyr, and Arg do not absorb light at this wavelength. Likewise, the even-electron fragment ions at m/z 551 and 160 were transparent at 355 nm and did not undergo further photodissociation. The slight decrease of the m/z 160 relative intensity at long trapping times needed to accommodate multiple laser pulses (Supplementary Figure S4a–d) is probably caused by a less efficient trapping at the edge of the LTQ stability region corresponding to 160/552 = 0.29 of the selected precursor ion m/z.
Figure 2

UVPD-MS3 mass spectra of hydrogen-rich peptide cation-radicals (m/z 552) generated by ETD of crown-ether complexes: (a) [AAAYR + 2H]+●, (b) [AAYAR + 2H]+●, (c) [AYAAR + 2H]+●, (d) [YAAAR + 2H]+●

UVPD showed a substantial depletion of the precursor cation radical populations after one laser pulse that exceeded 50% for all four peptide ions. However, all of them showed populations of residual photo-inactive cation radicals that did not undergo dissociation, asymptotically converging to 23%, 23%, 16%, and 6% for [AAAYR + 2H]+●, [AAYAR + 2H]+●, [AYAAR + 2H]+●, and [YAAAR + 2H]+●, respectively (Supplementary Figure S4a–d). Hence, the pulse-dependent experiments indicated that the populations of each of these cation radicals were not homogeneous and contained isomers of different light absorption properties.

The valine peptide analogues showed a similar behavior. UVPD resulted in a loss of H as the predominant fragment ion (Supplementary Figure S5a–d). Formation of the [z 1 + 2H]+ ion, albeit expected, could not be confirmed because the ion’s m/z was below the low-mass cutoff of the LTQ, 160/580 = 0.276. UVPD of [YAAVR + 2H]+● also produced a minor fragment by loss of C7H6O from the Tyr side chain. The pulse dependence of the charge-reduced AVAYR, AAYVR, AYAVR, and YAAVR cation-radicals (Supplementary Figure S6a–d) showed bimodal behavior consisting of rapid exponential depletion of the precursor ion within four laser pulses and leaving a substantial fraction of photo-inactive ions. The nature of the photoactive and photo-inactive isomers will be discussed later in the text.

Hydrogen-Deficient Peptide Cation Radicals

Peptide cation radicals of the hydrogen-deficient type were generated by CID of [Cu(tpy)(peptide)]2+● complexes, according to the previously reported method [6]. The hydrogen-deficient peptide cation radicals are denoted as [M]+● to distinguish them from the hydrogen-rich analogues. Further collisional activation of [M]+● was accomplished under a variety of conditions, including slow heating by resonant excitation in 3D and linear ion traps and acceleration of a mass selected ion beam in a tandem quadrupole mass spectrometer. These CID experiments yielded qualitatively similar results; the LTQ spectra are presented and discussed in the main text, the other data are in the Supplement (Supplementary Figure S7a–d). Ion assignments in the CID spectra were corroborated by accurate mass measurements (Supplementary Table S2).

CID of [M]+● produced MS3 spectra that showed dependence on the position of the Tyr residue in the peptide sequence (Fig. 3a–d). As the Tyr residue was moved toward the N-terminus, CID loss of C7H6O from the Tyr side chain formed fragment ions of increasing prominence that amounted to 8%, 39%, 75%, and 77% of the total ion intensity for [AAAYR]+●, [AAYAR]+●, [AYAAR]+●, and [YAAAR]+●, respectively. The loss of C7H6O is usually associated with the presence of a Tyr O-radical [7], and a recent UV-action spectroscopy study has identified a Tyr O-radical moiety in [YAAAR]+● [43]. Sequence fragment ions were observed for [AAAYR]+● where the [y 4 + 2H]+ ion (m/z 480) was a dominant product, accompanied by ions of the z-series (m/z 159, 322, 392, and 464). It is of note that some fragment ions of the c and z series from [AAAYR]+● are exactly isobaric; for example, both the [c 4 ]+ and [z 3 ]+ ions have the same theoretical m/z of 392.1928 for C18H26N5O5, consistent with the experimental m/z (Supplementary Table S2). A definite assignment of the fragment ion type was achieved by considering mass shifts in the homologous series, [AVAYR]+● and [AAAYR]+●, where there is no shift in the [z 2 ]+ (m/z 321) and [z 3 ]+ (m/z 392) ion masses (Supplementary Figure S8a–d), whereas a 28 Da shift would be expected for the equivalent [c 3 ]+ and [c 4 ]+ ions. Sequence ions of the z type were also observed for [AAYAR]+● (e.g., [z 2 + H]+●, [z 3 ]+, and [z 4 + H]+●) that were assigned by appropriate mass shifts in the spectrum of [AAYVR]+● (Supplementary Figure S8b).
Figure 3

CID-MS3 mass spectra of hydrogen-deficient peptide cation-radicals (m/z 550) generated from Cu(tpy) complexes: (a) [AAAYR]+●, (b) [AAYAR]+●, (c) [AYAAR]+●, (d) [YAAAR]+●

UVPD of [M]+● showed some novel features. All sequence variants of [M]+● were photoactive at 355 nm, pointing to radical-associated chromophores (Fig. 4a–d) [34]. UVPD of [AAAYR]+● and [AAVYR]+● showed dominant loss of H, which was accompanied by minor loss of C3H4NO and formation of the [y 4 + 2H]+ fragment ions. Upon moving the Tyr residue to the next position in [AAYAR]+● and [AAYVR]+●, photodissociation resulted in loss of H and C7H6O. Loss of C7H6O was dominant in the UVPD spectra of [AYAAR]+● and [YAAAR]+● (Fig. 4c, d) as well as in the spectra of their valine homologues (Supplementary Figure S9a–d). In addition to differences in the photofragmentation patterns, the isomeric peptide cation radicals also differed in the photodepletion efficiencies, as illustrated by the pulse-dependent UVPD spectra (Supplementary Figure S10a–d). Thus, [YAAAR]+● showed the most efficient photodepletion of the precursor ion at 50% after the first laser pulse (Supplementary Figure S10d). The main primary product ion, [Gα AAAR]+ undergoes further photodissociation by loss of H and forming the [y 4 ]+ fragment ion as the main sequence product. Note that [y 4 ]+ in the current notation corresponds to a (y 4 − 2H)+ species by the previously used nomenclature and represents a common photodissociation product of peptide Cα-radical ions [34]. The formation of [AGα AAR]+ and [AAGα AR]+ intermediates is also seen in the photodepletion curves of the pertinent [AYAAR]+● and [AAYAR]+● ions (Supplementary Figure S10b, c) and their Val homologues (Supplementary Figure S11a–d). The isomeric Gly Cα-radical ions (m/z 444) were further investigated by CID and UVPD.
Figure 4

UVPD-MS3 mass spectra of hydrogen-deficient peptide cation-radicals (mnz 550) generated from Cu(tpy) complexes: (a) [AAAYR]+●, (b) [AAYAR]+●, (c) [AYAAR]+●, (d) [YAAAR]+●

Effects of Gly Cα Radical Position

The Gly Cα radicals were generated by CID-MS3 of [M]+●, isolated by mass and subjected to CID-MS4 and UVPD-MS4. The UVPD-MS4 spectra showed dissociations that were highly specific of the position of the Gly Cα radical site (Fig. 5a–d). Starting with [AAAGα R]+, UVPD resulted in the formation of [y 2 ]+ (m/z 230, GR) and [y 3 ]+ (m/z 301, AGR) sequence fragment ions in addition to loss of H, which was another abundant dissociation channel (Fig. 5a). The formation of the [y 2 ]+ ion proceeds by β-fission of the CO−NH bond adjacent to the Glyα radical and does not require a hydrogen migration. To form the [y 3 ]+ ion, the Hα from the adjacent Ala residue must migrate to form an Alaα radical that promotes CO−NH bond dissociation at the adjacent position [34]. Hydrogen atom migration in [AAAGα R]+ to the Gα radical from the proximate Arg Cα or Cβ positions can also be considered to explain the loss of H forming a product with ion an α,β-dehydro-Arg moiety. Loss of H is either absent or much less abundant in the other isomers where the Gα radical is flanked by Ala residues. The laser pulse-dependent measurements of [AAAGα R]+ (Supplementary Figure S12a) indicated an exponential depletion of the [AAAGα R]+ ion intensity, whereas the [y 2 ] to [y 3 ] abundance ratio was essentially constant at 4.1:1.
Figure 5

UVPD-MS4 mass spectra of (a) [AAAGα R]+, (b) [AAGα AR]+, (c) [AGα AAR]+, (d) [Gα AAAR]+

UVPD of the [AAGα AR]+ ion was even more specific, inducing CO–NH bond cleavage in the position adjacent to the Glyα radical and producing the [y 3 ]+ ion (m/z 301, GAR) as the major product (Fig. 5b). Noteworthy is the absence of loss of H in this case. These features are reflected by the photodepletion curve that shows rapid decrease of the [AAGα AR]+ relative intensity and formation of the [y 3 ]+ (GAR) fragment ion (Supplementary Figure S12b).

UVPD of [AGα AAR]+ also showed substantial specificity in breaking the CO−NH bond adjacent to the Gα radical and forming the [y 4 ]+ ion (m/z 372, GAAR) (Fig. 5c). As a side reaction, Ala Hα atom migration moved the radical and triggered CO−NH bond dissociation between the Gly and Ala residues to produce the [y 3 ]+ (AAR) fragment ion at m/z 315. The overall photodepletion curve for AGα AAR]+ showed an exponential decay resulting in virtually complete dissociation after seven laser pulses (Supplementary Figure S12c).

Finally, UVPD of [Gα AAAR]+ was the least efficient of the four isomers (Supplementary Figure S12d) and resulted in loss of H, C2H2NO, and C2H4NO neutral molecules (Fig. 5d). These minor backbone fragmentations indicate an H atom migration to and from the adjacent Ala residue, followed by CO−NH bond dissociations forming the [y 4 + 2H]+ and [y 4 ]+ ions. Loss of H can proceed by β-fission from the N-terminal amine group without need for H-atom migration.

In contrast to UVPD, CID-MS4 was much less specific of the radical position. The CID-MS4 spectra (Fig. 6a–d) show the dominant formation of the [y 4 + 2H]+ ions from all four isomers, appearing at m/z 374 for [AAAGα R]+, [AAGα AR]+, [AGα AAR]+, and m/z 388 from [Gα AAAR]+, indicating hydrogen atom migrations and preferential cleavage of the CO−NH bond between the N-terminal and adjacent residues. In spite of this dominant general feature, the CID-MS4 spectra show some differences in the fragment on relative intensities. A conspicuous feature is the formation of even-electron [z 1 ]+-[z 3 ]+ ions by N—Cα bond cleavage accompanied by H atom migration onto the neutral fragment. In contrast, N—Cα cleavage of the bond connecting the N-terminal residue leads to [z 4 + H]+● cation-radical fragments in all sequences. In the absence of more detailed mechanistic investigation, these effects are difficult to relate to the ion structure.
Figure 6

CID-MS4 mass spectra of (a) [AAAGα R]+, (b) [AAGα AR]+, (c) [AGα AAR]+, (dn [Gα AAAR]+

Mechanistic Study of Hydrogen Loss

The prominent loss of H upon UVPD of [AAAYR]+● and its virtual absence in CID raised the question of the ion structure and origin of the hydrogen atom. To address these topics, we resorted to use deuterium labeling in a smaller homologue, [AAYR]+●, which showed a quite analogous behavior upon CID and UVPD, namely, abundant photo-induced loss of H, (Supplementary Figures S13, S14a), indicating that the main structure and reactivity features of [AAYR]+● were similar to those governing the dissociation of [AAAYR]+●. The advantage of using [AAYR]+● was due to its lower number of exchangeable protons in H/D exchange experiments. UVPD of completely H/D exchanged [d 11-AAYR]+● showed a highly specific (94%) loss of D from one of the exchangeable positions (Supplementary Figure S14b). However, distinction among hydrogen atoms in the exchangeable positions is a daunting task that has been achieved using special techniques [44, 45] that are not applicable to peptide ions. An alternative method is to block by methylation a specific position in the ion, as previously applied in a study of peptide electron transfer dissociations [46]. To this end, we synthesized a AAY(OCH3)R peptide in which the Tyr residue was replaced by O-methyl tyrosine, thus blocking the phenol hydroxyl group. UVPD of the [AAY(OCH3)R]+● ion (m/z 493, Supplementary Figure S15a) showed abundant loss of H in contrast to CID of the ion that generated backbone [z 1 + H]+●, [z 2 ]+, and [y 3 + 2H]+ fragment ions, as well as an ion from CO2 loss (Supplementary Figure S16). H/D exchange in [AAY(OMe)R]+● produced a d 10 isotopologue (m/z 503), which upon UVPD showed a major loss of D forming the fragment ion at m/z 501 (Supplementary Figure S15b). These experiments indicated that the photo-induced loss of H from [AAYR]+●, and, by implication also from [AAAYR]+●, can originate from exchangeable positions other than the Tyr hydroxyl. It should be noted, however, that methylation of the Tyr residue changes its electronic and chemical properties in addition to blocking the phenol hydroxyl. This is indicated by the UVPD spectrum of [AAY(OMe)R]+●, which shows a specific formation of the [y 1 ]+ fragment ion (m/z 158, Supplementary Figure S15a), which is much less abundant when produced from [AAYR]+● (Supplementary Figure S14a). A pulse dependence experiment showed exponential depletion of the [AAY(OCH3)R]+● intensity (Supplementary Figure S17), indicating that all the components of the ion population absorbed light at 355 nm. We note that the absorption spectra of peptide cation radicals depend on the position of the Cα radical and ion conformation [34, 43], and elucidating this effect would require a dedicated study.

Discussion

The experimental data using CID and UVPD showed distinct features that can be related to the nature of the peptide cation radicals and the mode of activation. It is useful to first reiterate the differences in the activation modes because they apply to both hydrogen-rich and -deficient peptide cation radicals. CID in the ion trap is performed by resonant translational excitation of the mass-selected ion under slow heating conditions [47], which upon collisions with the bath gas (He at 3 mTorr in the LTQ) results in vibrational excitation of the ground electronic state of the precursor cation radical. The fragment ions are not accelerated and their collisions with the bath gas result in cooling that occurs on the 10–20 ms time scale [48]. In contrast, UVPD is a “sudden” excitation method that generates an electronically excited state of the peptide ion with an internal energy (Eint) given by the sum of the photon energy (3.493 eV, 337 kJ mol–1) and the thermal rovibrational energy (H rovib) of the ion at the ion trap temperature. For [AAAYR + 2H]+● and related ions, H rovib has a mean value of 105 kJ mol–1 at 310 K, giving Eint = 337 + 105 = 442 kJ mol–1. The photoexcited peptide ion can dissociate on the same potential energy surface, utilizing the electronic part of the excitation energy, or undergo vibronic conversion to a vibrationally excited ground electronic state with Eint. Both these processes compete with collisional de-excitation of the excited electronic state and vibrational cooling of the hot ground state.

Starting with the hydrogen-rich cation radicals, both the CID and UVPD spectra indicated that the stable, long-lived cation radicals were distinctly different from the reactive intermediates produced by electron attachment to peptide dications. This raises the question of the cation radical structure and formation upon ETD of the CE complexes. Crown ethers bind to charged groups by forming strong hydrogen bonds in gas-phase ions [49]. The ETD spectra of the doubly charged peptide–CE complexes indicate rather nonspecific binding to the N-terminal ammonium and Arg guanidinium charged groups. This finding is at odds with the bonding energies of 18-crown-6-ether to singly protonated amino acids, reported by Chen and Rodgers [50], where N-terminal bonding was found to be stronger than side-chain bonding in arginine. The probable reason for this apparent discrepancy is that the competitive binding of 18-crown-6-ether to a charged group in a doubly protonated peptide disrupts this group’s internal solvation, and this loss of attractive interactions can compensate for the the stronger binding to the ligand. In addition, disruption of internal solvation of the charged group by the peptide amide groups affects the ion conformation [34].

Another large effect, which is related to electron transfer, is that coordination with CE substantially lowers the intrinsic recombination energy of the charged group, for example, from 4.31 eV in isolated CH3NH3 + to 1.74 eV in the CH3NH3 +–CE complex [51]. The calculated recombination energy of the ethylguanidinium cation also showed a drop from 380 kJ mol–1 (3.94 eV) in the free ion to 231 kJ mol–1 (2.40 eV) in the CE complex (Table S3), illustrating the effect of CE coordination on the electronic structure of the cation and radical.

Charge reduction by electron attachment to the charged group substantially lowers its CE bonding energy; for example, from 273 kJ mol–1 in the CH3NH3 +–CE complex to 35 kJ mol–1 in its charge-reduced counterpart [51]. Similar effects were indicated by calculations of N-ethylguanidinium ion and radical CE complexes that stand for the Arg–CE bonding (Supplementary Table S3). The 0 K bonding energy to CE was calculated to decrease from ΔH 0,diss = 197 kJ mol–1 in the ion to 56 kJ mol–1 in the radical. The calculated CE bonding free energy for ethylguanidinium radical at the experimental temperature of 310 K, ΔG 310,diss = 16 kJ mol–1, indicated that binding of the CE ligand to the charge-reduced Arg guanidinium as well as N-terminal ammonium functional groups is weak and, hence, electron transfer targeting the coordinated charged group is expected to result in a facile loss of the CE ligand.

The peptide cation radicals resulting from electron attachment and CE loss have the odd electron in the charge-reduced group, which is an ammonium radical at the N-terminus or a guanidinium radical in the Arg side chain. This electron distribution is fundamentally different from that accessed by direct electron attachment to the isolated peptide dication, where the reactive electronic states are charge-stabilized amide π* states [52, 53]. This interpretation is consistent with the stark differences in the peptide cation radical reactivity depending on their mode of formation. The isomeric charge-reduced cation radicals from peptide–CE complexes also have different stabilities, whereby the ammonium radicals are only weakly bound and can rapidly dissociate by competing losses of H and ammonia [54]. This may account for the abundant [M + 2H−CE−NH3]+● fragment ions in the ETD spectra of the complexes (m/z 535, Supplementary Figure S2a–d). In contrast, arginine guanidinium radicals are intrinsically stable [45, 55]. Vibrational excitation, as in CID, results in competitive loss of H and guanidine [56], accounting for the pertinent fragment ions, m/z 551 and 493, respectively, in the CID spectra (Fig. 1). Arginine radicals formed by electron transfer have been shown to be moderately efficient hydrogen atom acceptors [55, 57]. This may account for the formation of the [z 1 + 2H]+ fragment ions, as sketched for [AAYAR + 2H]+● in Scheme 1, as well as loss of C7H6O, which is triggered by transfer of the phenol hydrogen. Detailed mechanisms of the hydrogen transfer and bond cleavage steps leading to these ions have not been established yet.
Scheme 1

Proposed reaction sequence for the loss of H and formation of [z 1 + 2H]+ fragment ions

Guanidinium radicals are strong chromophores absorbing light at 320, 341, and 357 nm, with the corresponding oscillator strength factors of 0.07, 0.18, and 0.01 [58]. This is consistent with the efficient photodepletion of the charge-reduced hydrogen-rich cation radicals at 355 nm (Supplementary Figure S4a–d). Excited states of arginine radicals have been shown to undergo extremely fast loss of hydrogen [56], which is consistent with the dominant photodissociation channels in the UVPD spectra.

The 6%–23% fractions of photo-inactive hydrogen-rich peptide cation radicals (Supplementary Figure S4a–d) belong to ion structures that are difficult to directly assign and likely differ for the sequence isomers. Based on the analysis of peptide radical chromophores [58], dihydrophenol radicals produced by H-atom migration to the Tyr side chain, and [a + x]+● ion–molecule complexes are possible candidates for structures not absorbing at 355 nm.

The hydrogen-deficient [M]+● cation radicals show collision-induced and photodissociations that depend on both the activation mode and position of the Tyr residue in the peptide sequence. CID and UVPD of [YAAAR]+● are most straightforward in that they both result in a dominant loss of the Tyr side chain. This is consistent with the Tyr-O radical structure of this ion where loss of C7H6O is the lowest energy process in CID and the Tyr-O radical is the absorbing chromophore for UVPD [43]. We cannot distinguish whether UVPD occurs from an excited electronic state or after vibronic transition to a vibrationally hot ground electronic state because both are expected to display similar reactivity. In contrast, the other peptide sequences show substantial differences between CID and UVPD, indicating large differences in the excited-state and ground-state reactivity. With [AAAYR]+●, CID induces hydrogen atom migrations forming intermediates that dissociate by backbone cleavages and loss of C7H6O, forming the various fragment ions seen in the spectrum. In contrast, UVPD proceeds by only two major channels, one being loss of protogenic exchangeable H and the other a backbone cleavage leading to the [y 4 + 2H]+ ion. The latter reaction can be interpreted as proceeding from a hot vibrational state accessed by vibronic transition from the excited electronic state. In contrast, the loss of H does not have an analogy in CID and is likely proceeding from an excited electronic state of the ion. A previous UV photodissociation action spectroscopy study has concluded that [AAAYR]+● was a mixture of isomeric radicals [43], and thus it is possible that the observed photodissociations originated from different radical isomers.

Conclusions

The results reported in this comparative study allow us to arrive at the following conclusions.

Both the hydrogen-rich peptide cation radicals produced by electron-transfer reduction and their hydrogen-deficient counterparts produced by electron-transfer oxidation contain chromophores associated with the radical moieties that result in photodissociation at 355 nm. The hydrogen-rich peptide cation radicals contain major fractions of structures with Arg guanidinium radical groups. Consequently, both collision-induced dissociation and photodissociation of the long-lived hydrogen-rich cation radicals are diametrically different from electron-transfer dissociation of the corresponding peptide dications. CID and UVPD of hydrogen-deficient peptide cation radicals show dependence on the position on the Tyr residue. Ions in which the Tyr residue is in sequence positions remote from the charged Arg group show larger proportions of Tyr-O radicals that undergo collision-induced and photodissociative loss of C7H6O from the Tyr side chain.

Notes

Acknowledgments

Research at University of Washington received support from the Chemistry Division of the National Science Foundation (grant CHE-1359810). I.K.C. thanks support from the Hong Kong Research Grants Council (project nos. HKU 701613P and 173306015), the University of Hong Kong (Seed Funding Program for Basic Research 201411159067 and 201511159023); X.Y.M. thanks the Hong Kong RGC for supporting studentship. F.T. thanks the Royal Society Kan Tong Po Professorship for supporting his stay at the University of Hong Kong in September–December 2014. Support from the Klaus and Mary Ann Saegebarth Endowment is gratefully acknowledged.

Supplementary material

13361_2016_1425_MOESM1_ESM.pdf (3.8 mb)
ESM 1 (PDF 3932 kb)

References

  1. 1.
    Tureček, F., Julian, R.R.: Peptide radicals and cation-radicals in the gas phase. Chem. Rev. 113, 6691–6733 (2013)CrossRefGoogle Scholar
  2. 2.
    Grotemeyer, J., Boesl, U., Walter, K., Schlag, E.W.: A general soft ionization method for mass spectrometry: resonance-enhanced multiphoton ionization of biomolecules. Org. Mass Spectrom. 21, 645–653 (1986)CrossRefGoogle Scholar
  3. 3.
    Grotemeyer, J., Schlag, E.W.: Peptides investigated by laser desorption-multiphoton ionization mass spectrometry.Part VII. Org. Mass Spectrom. 23, 388–396 (1988)CrossRefGoogle Scholar
  4. 4.
    Grotemeyer, J., Schlag, E.W.: Biomolecules in the gas phase: multiphoton ionization mass spectrometry. Acc. Chem. Res. 22, 399–406 (1989)CrossRefGoogle Scholar
  5. 5.
    Chu, I.K., Rodriquez, C.F., Lau, T.C., Hopkinson, A.C., Siu, K.W.M.: Molecular radical cations of oligopeptides. J. Phys. Chem. B 104, 3393–3397 (2000)CrossRefGoogle Scholar
  6. 6.
    Chu, I.K., Lam, C.N.W.: Generation of peptide radical dications via low energy collision-induced dissociation of [Cu(II)(terpy)(MþH)]3þ. J. Am. Soc. Mass Spectrom. 16, 1795–1804 (2005)CrossRefGoogle Scholar
  7. 7.
    Barlow, C.K., Wee, S., McFadyen, W.D., O’Hair, R.A.J.: Designing copper(II) ternary complexes to generate radical cations of peptides in the gas phase: Role of the auxiliary ligand. Dalton Trans. 3199–3204 (2004)Google Scholar
  8. 8.
    Hopkinson, A.C., Siu, K.W.M.: Peptide radical cations. In: Laskin, J., Lifshitz, C., (eds.) Principles of Mass Spectrometry Applied to Biomolecules. Wiley-Interscience, Chap 9, pp. 301–336 (2006)Google Scholar
  9. 9.
    Chu, I.K., Laskin, J.: Formation of peptide radical ions through dissociative electron transfer in ternary metal-ligand-peptide complexes. Eur. J. Mass Spectrom. 17, 543–556 (2011)CrossRefGoogle Scholar
  10. 10.
    Hodyss, R., Cox, H.A., Beauchamp, J.L.: Bioconjugates for tunable peptide fragmentation: Free radical initiated peptide sequencing (FRIPS). J. Am. Chem. Soc. 127, 12436–12437 (2005)CrossRefGoogle Scholar
  11. 11.
    Ly, T., Julian, R.R.: Residue-specific radical-directed dissociation of whole proteins in the gas phase. J. Am. Chem. Soc. 130, 351–358 (2008)CrossRefGoogle Scholar
  12. 12.
    Zubarev, R.A., Kelleher, N.L., McLafferty, F.W.: Electron capture dissociation of multiply charged protein cations. A nonergodic process. J. Am. Chem. Soc. 120, 3265–3266 (1998)CrossRefGoogle Scholar
  13. 13.
    Chakraborty, T., Holm, A.I.S., Hvelplund, P., Nielsen, S.B., Poully, J.-C., Worm, E.S., Williams, E.R.: On the survival of peptide cations after electron capture: role of internal hydrogen bonding and microsolvation. J. Am. Soc. Mass Spectrom. 17, 1675–1680 (2006)CrossRefGoogle Scholar
  14. 14.
    Holm, A.I.S., Hvelplund, P., Kadhane, U., Larsen, M.K., Liu, B., Nielsen, S.B., Panja, S., Pedersen, J.M., Skrydstrup, T., Støchkel, K., Williams, E.R., Worm, E.S.: On the mechanism of electron-capture-induced dissociation of peptide dications from 15N-labeling andcrown-ether complexation. J. Phys. Chem. A 111, 9641–9653 (2007)CrossRefGoogle Scholar
  15. 15.
    Jensen, C.S., Wyer, J.A., Houmøller, J., Hvelplund, P., Nielsen, S.B.: Electron-capture induced dissociation of doubly charged dipeptides: on the neutral losses and N-Cα bond cleavages. Phys. Chem. Chem. Phys. 13, 18373–8 (2011)CrossRefGoogle Scholar
  16. 16.
    Turecek, F., Yao, C., Fung, Y.M.E., Hayakawa, S., Hashimoto, M., Matsubara, H.: Histidine-containing radicals in the gas phase. J. Phys. Chem. B 113, 7347–7366 (2009)CrossRefGoogle Scholar
  17. 17.
    Hayakawa, S., Hashimoto, M., Matsubara, H., Turecek, F.: Dissecting the proline effect: dissociations of proline radicals formed by electron transfer to protonated Pro-Gly and Gly-Pro dipeptides in the gas phase. J. Am. Chem. Soc. 129, 7936–7949 (2007)CrossRefGoogle Scholar
  18. 18.
    Byskov, C.S., Nielsen, S.B.: On the formation, stability, and dissociation of peptide radicals after femtosecond electron transfer from alkali metal atoms. Int. J. Mass Spectrom. 390, 2–13 (2015)CrossRefGoogle Scholar
  19. 19.
    Syka, J.E.P., Coon, J.J., Schroeder, M.J., Shabanowitz, J., Hunt, D.F.: Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 101, 9528–9533 (2004)CrossRefGoogle Scholar
  20. 20.
    Biemann, K.: Contributions of mass spectrometry to peptide and protein structure. Biomed. Environ. Mass Spectrom. 16, 99–111 (1988)CrossRefGoogle Scholar
  21. 21.
    Ly, T., Yin, S., Loo, J.A., Julian, R.R.: Electron-induced dissociation of protonated peptides yields backbone fragmentation consistent with a hydrogen-deficient radical. Rapid Commun. Mass Spectrom. 23, 2099–2101 (2009)CrossRefGoogle Scholar
  22. 22.
    Sun, Q., Nelson, H., Ly, T., Stoltz, B.M., Julian, R.R.: Side chain chemistry mediates backbone fragmentation in hydrogen-deficient peptide radicals. J. Proteome Res. 8, 958–966 (2009)CrossRefGoogle Scholar
  23. 23.
    Chung, T.W., Tureček, F.: Backbone and side-chain specific dissociations of z Ions from non-tryptic peptides. J. Am. Soc. Mass Spectrom. 21, 1279–1295 (2010)CrossRefGoogle Scholar
  24. 24.
    Moss, C.L., Chung, T.W., Wyer, J.A., Nielsen, S.B., Hvelplund, P., Tureček, F.: Dipole-guided electron capture causes abnormal dissociations of phosphorylated pentapeptides. J. Am. Soc. Mass Spectrom. 22, 731–751 (2011)CrossRefGoogle Scholar
  25. 25.
    Chung, T.W., Tureček, F.: Proper and improper aminoketyl radicals in electron-based peptide dissociations. Int. J. Mass Spectrom. 301, 55–61 (2011)CrossRefGoogle Scholar
  26. 26.
    Xu, M., Song, T., Quan, Q., Hao, Q., Fang, D.-C., Siu, C.-K., Chu, I.K.: Effect of the N-terminal basic residue on facile Cα–C bond cleavages of aromatic-containing peptide radical cations. Phys. Chem. Chem. Phys. 13, 5888–5896 (2011)CrossRefGoogle Scholar
  27. 27.
    Ledvina, A.R., Chung, T.W., Hui, R., Coon, J.J., Tureček, F.: Cascade dissociations of peptide cation-radicals. Part 2. Infrared multiphoton dissociation and mechanistic studies of z-ions from pentapeptides. J. Am. Soc. Mass Spectrom. 23, 1351–1363 (2012)CrossRefGoogle Scholar
  28. 28.
    Hao, Q., Song, T., Ng, D.C.M., Quan, Q., Siu, C.-K., Chu, I.K.: Arginine-facilitated isomerization: radical-induced dissociation of aliphatic radical cationic glycylarginyl(iso)leucine tripeptides. J. Phys. Chem. B 116, 7627–7634 (2012)CrossRefGoogle Scholar
  29. 29.
    Zhao, J., Song, T., Xu, M., Quan, Q., Siu, K.W.M., Hopkinson, A.C., Chu, I.K.: Intramolecular hydrogen atom migration along the backbone of cationic and neutral radical tripeptides and subsequent radical-induced dissociations. Phys. Chem. Chem. Phys. 14, 8723–8731 (2012)CrossRefGoogle Scholar
  30. 30.
    Zhang, X., Julian, R.R.: Exploring radical migration pathways in peptides with positional isomers, deuterium labeling, and molecular dynamics simulations. J. Am. Soc. Mass Spectrom. 24, 524–533 (2013)CrossRefGoogle Scholar
  31. 31.
    Thomas, D.A., Sohn, C.H., Gao, J., Beauchamp, J.L.: Hydrogen bonding constrains free radical reaction dynamics at serine and threonine residues in peptides. J. Phys. Chem. A 118, 8380–8392 (2014)CrossRefGoogle Scholar
  32. 32.
    Xu, M., Tang, W.-K., Mu, X., Ling, Y., Siu, C.-K., Chu, I.K.: α-Radical-induced CO2 loss from the aspartic acid side chain of the collisional induced tripeptide aspartylglycylarginine radical cation. Int. J. Mass Spectrom. 390, 56–62 (2015)CrossRefGoogle Scholar
  33. 33.
    Mu, X.Y., Song, T., Xu, M., Lai, C.K., Siu, C.K., Laskin, J., Chu, I.K.: Discovery and mechanistic studies of facile N-terminal Cα–C bond cleavages in the dissociation of tyrosine-containing peptide radical cations. J. Phys. Chem. B 118, 4273 (2014)CrossRefGoogle Scholar
  34. 34.
    Shaffer, C.J., Marek, A., Pepin, R., Slováková, K., Tureček, F.: Combining UV photodissociation with electron transfer for peptide structure analysis. J. Mass Spectrom. 50, 470–475 (2015)CrossRefGoogle Scholar
  35. 35.
    Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G.A., Nakatsuji, H., Caricato, M., Li, X., Hratchian, H.P., Izmaylov, A.F., Bloino, J., Zheng, G., Sonnenberg, J.L., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery Jr., J.A., Peralta, J.E., Ogliaro, F., Bearpark, M., Heyd, J.J., Brothers, E., Kudin, K.N., Staroverov, V.N., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J.C., Iyengar, S.S., Tomasi, J., Cossi, M., Rega, N., Millam, J.M., Klene, M., Knox, J.E., Cross, J.B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R.E., Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W., Martin, R.L., Morokuma, K., Zakrzewski, V.G., Voth, G.A., Salvador, P., Dannenberg, J.J., Dapprich, S., Daniels, A.D., Farkas, O., Foresman, J.B., Ortiz, J.V., Cioslowski, J., Fox, D.J.: Gaussian 09, Revision A.02. Gaussian, Inc, Wallingford CT (2009)Google Scholar
  36. 36.
    Becke, A.D.: A new mixing of Hartree-Fock and local density-functional theories. J. Chem. Phys. 98, 1372–1377 (1993)CrossRefGoogle Scholar
  37. 37.
    Zhao, Y., Truhlar, D.G.: The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals. Theor. Chem. Accounts 120, 215–241 (2008)CrossRefGoogle Scholar
  38. 38.
    Chai, J.D., Head-Gordon, M.: Long-range corrected hybrid density functionals with damped atom-atom dispersion corrections. Phys. Chem. Chem. Phys. 10, 6615–6620 (2008)CrossRefGoogle Scholar
  39. 39.
    Møller, C., Plesset, M.S.: A note on an approximation treatment for many-electron systems. Phys. Rev. 46, 618–622 (1934)CrossRefGoogle Scholar
  40. 40.
    Chu, I.K., Siu, C.-K., Lau, J.K.-C., Tang, W.K., Mu, X., Lai, C.K., Guo, X., Wang, X., Li, N., Yao, Z., Xia, Y., Kong, X., Oh, H.-B., Ryzhov, V., Tureček, F., Hopkinson, A.C., Siu, K.W.M.: Proposed nomenclature for peptide ion fragmentation. Int. J. Mass Spectrom. 390, 24–27 (2015)CrossRefGoogle Scholar
  41. 41.
    Marek, A., Pepin, R., Peng, B., Laszlo, K.J., Bush, M.F., Tureček, F.: Electron transfer dissociation of photolabeled peptides. Backbone cleavages compete with diazirine ring rearrangements. J. Am. Soc. Mass Spectrom. 24, 1641–1653 (2013)CrossRefGoogle Scholar
  42. 42.
    Ehlerding, A., Jensen, C.S., Wyer, J.A., Holm, A.I.S., Jørgensen, P., Kadhane, U., Larsen, M.K., Panja, S., Poully, J.C., Worm, E.S., Zettergren, H., Hvelplund, P., Brøndsted Nielsen, S.: Influence of temperature and crown ether complex formation on the charge partitioning between z and c fragments formed after electron capture by small peptide dications. Int. J. Mass Spectrom. 282, 21–27 (2009)CrossRefGoogle Scholar
  43. 43.
    Viglino. E., Shaffer, C.J., Tureček, F.: UV-VIS action spectroscopy and structures of tyrosine peptide cation radicals in the gas phase. Angew. Chem. Int. Ed. Engl. 55, (2016). doi:  10.1002/anie.201602604
  44. 44.
    Gerbaux, P., Tureček, F.: Protonated carbonic acid and the trihydroxymethyl radical in the gas phase. A neutralization-reionization mass spectrometric and ab initio/RRKM study. J. Phys. Chem. A 106, 593–5950 (2002)CrossRefGoogle Scholar
  45. 45.
    Hao, C., Seymour, J.L., Tureček, F.: Electron super-rich radicals in the gas phase. A neutralization-reionization mass spectrometric and ab initio/RRKM study of diaminohydroxymethyl and triaminomethyl radicals. J. Phys. Chem. A 111, 8829–8843 (2007)CrossRefGoogle Scholar
  46. 46.
    Crizer, D.M., McLuckey, S.A.: Electron transfer dissociation of amide nitrogen methylated polypeptide cations. J. Am. Soc. Mass Spectrom. 20, 1349–1354 (2009)CrossRefGoogle Scholar
  47. 47.
    McLuckey, S.A., Goeringer, D.E.: Slow heating methods in tandem mass spectrometry. J. Mass Spectrom. 32, 461–474 (1997)CrossRefGoogle Scholar
  48. 48.
    Pepin, R., Turecek, F.: Kinetic ion thermometers for electron transfer dissociation. J. Phys. Chem. B 119, 2818–2826 (2015)CrossRefGoogle Scholar
  49. 49.
    Julian, R.R., Beauchamp, J.: Site-specific sequestering and stabilization of charge in peptides by supramolecular adduct formation with 18-crown-6 ether by way of electrospray ionization. Int. J. Mass Spectrom. 210/211, 613–623 (2001)CrossRefGoogle Scholar
  50. 50.
    Chen, Y., Rodgers, M.T.: Structural and energetic effects in the molecular recognition of amino acids by 18-crown-6. J. Am. Chem. Soc. 134, 5863–5875 (2012)CrossRefGoogle Scholar
  51. 51.
    Holm, A.I.S., Larsen, M.K., Panja, S., Hvelplund, P., Nielsen, S.B., Leib, R.D., Donald, W.A., Williams, E.R., Hao, C., Tureček, F.: Electron capture, femtosecond electron transfer and theory: A study of noncovalent crown ether 1, n-diammonium alkane complexes. Int. J. Mass Spectrom. 276, 116–126 (2008)CrossRefGoogle Scholar
  52. 52.
    Syrstad, E.A., Tureček, F.: Toward a general mechanism of electron-capture dissociation. J. Am. Soc. Mass Spectrom. 16, 208–224 (2005)CrossRefGoogle Scholar
  53. 53.
    Sobczyk, M., Anusiewicz, I., Berdys-Kochanska, J., Sawicka, A., Skurski, P., Simons, J.: Coulomb-assisted dissociative electron attachment: Application to a model peptide. J. Phys. Chem. A 109, 250–258 (2005)CrossRefGoogle Scholar
  54. 54.
    Yao, C., Tureček, F.: Hypervalent ammonium radicals. Competitive N–C and N–H bond dissociations in methylammonium and ethylammonium. Phys. Chem. Chem. Phys. 7, 912–920 (2005)CrossRefGoogle Scholar
  55. 55.
    Chen, X., Tureček, F.: The arginine anomaly: arginine radicals are poor hydrogen atom donors in electron transfer induced dissociations. J. Am. Chem. Soc. 128, 12520–12530 (2006)CrossRefGoogle Scholar
  56. 56.
    Moss, C.L., Liang, W., Li, X., Tureček, F.: The early life of a peptide cation radical. Ground and excited-state trajectories of electron-based peptide dissociations during the first 330 femtoseconds. J. Am. Soc. Mass Spectrom. 23, 446–459 (2012)CrossRefGoogle Scholar
  57. 57.
    Hayakawa, S., Matsubara, H., Panja, S., Hvelplund, P., Nielsen, S.B., Chen, X., Tureček, F.: Experimental evidence for an inverse hydrogen migration in arginine radicals. J. Am. Chem. Soc. 130, 7645–7654 (2008)CrossRefGoogle Scholar
  58. 58.
    Turecek, F.: Benchmarking Electronic excitation energies and transitions in peptide radicals. J. Phys. Chem. A 119, 10101–10111 (2015)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2016

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of WashingtonSeattleUSA
  2. 2.Department of ChemistryUniversity of Hong KongHong KongChina

Personalised recommendations