Letter pubs.acs.org/JPCL
UV Action Spectroscopy of Gas-Phase Peptide Radicals Huong T. H. Nguyen, Christopher J. Shaffer, Robert Pepin, and František Tureček* Department of Chemistry, University of Washington, Bagley Hall, Box 351700, Seattle, Washington 981195-1700, United States S Supporting Information *
ABSTRACT: UV photodissociation (UVPD) action spectroscopy is reported to provide a sensitive tool for the detection of radical sites in gas-phase peptide ions. UVPD action spectra of peptide cation radicals of the z-type generated by electron-transfer dissociation point to the presence of multiple structures formed as a result of spontaneous isomerizations by hydrogen atom migration. N-terminal Cα radicals are identified as the dominant components, but the content of isomers differing in the radical defect position in the backbone or side chain depends on the nature of the aromatic residue with phenylalanine being more prone to isomerization than tryptophan. These results illustrate that spontaneous hydrogen atom migrations can occur in peptide cation-radicals upon electron-transfer dissociation.
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methods are needed to elucidate the structure of these fascinating radical intermediates. Peptide cation-radicals of both hydrogen-rich and hydrogendeficient types have recently been shown to carry radicalassociated chromophore groups absorbing light in the near-UV region,15 where the closed-shell peptide groups are transparent and do not interfere with analysis.16−18 Irradiation at 355 nm has been shown to induce specific photodissociations, which has allowed us to assign the radical site positions in peptide cation-radicals.15 We now extend the near-UV photodissociation (UVPD) of peptide cation radicals to full action spectroscopy in the 210−400 nm region, whereby multiple photodissociation channels are monitored as a function of the laser wavelength. The new experimental method is demonstrated with the structure analysis of z4+• ions generated in situ by electron-transfer dissociation (ETD) of doubly protonated AAWAR and AAFAR peptide ions. The ground electronic state chemistry of these z4+• ions, as probed by CID, consists of a single dominant channel (60%) involving transfer of a benzyl hydrogen atom, followed by radical-induced backbone cleavage of the adjacent Cα-CO bond (Scheme 1).13 We now apply UV action spectroscopy to probe if hydrogen migration occurs spontaneously in z4+• fragment ions produced by ETD. The UV action spectroscopy experiments are complemented by time-dependent density functional theory (TD-DFT) calculations of excited electronic states of several cation-radicals of interest. Ion Formation. The z4+• ions were generated by a standard procedure whereby precursor [peptide +2H]2+ dications (peptide = AAWAR or AAFAR) were produced by electrospray
adical-containing peptide and protein ions of various protonation types have recently become available thanks to multiple advances in experimental gas-phase ion chemistry.1 Such gas-phase systems deal with isolated molecules or structures and are complementary to condensed-phase investigations of radical intermediates in enzyme catalysis2 and radical-induced protein damage.3 Novel and electronically unusual species such as hydrogen-rich peptide cation radicals can be produced as transient intermediates by electron transfer to multiply charged peptide ions.4 Hydrogen-deficient peptide cation radicals can be generated by photodissociation5 or collision-induced dissociation6,7 of suitably tagged peptide ions. Further hydrogen deficiency can be achieved by yet another method where gas-phase peptide cation-radicals are generated utilizing intramolecular electron transfer to a ligated transition metal ion.8,9 Stoichiometrically, hydrogen-deficient peptide cation radicals formally correspond to peptide molecules missing an electron.1 Importantly, some of these types of gasphase peptide cation radicals are more accurately described as distonic ions,10,11 in which the charge and radical reside at distant, that is, nonvicinal and nongeminal, sites in the molecule. A classical example of distonic peptide cation radicals are z+• fragment ions being formed by N−Cα bond cleavage of peptide ions upon electron attachment (Scheme 1).12 z+• ions are typically represented as N-terminal deaminated peptide Cα radicals carrying the charging proton at a remote basic residue, which is often lysine or arginine. Although various z+• ions have been the subject of mechanistic studies dealing with dissociation energetics and kinetics,13 their structures remain uncertain. The uncertainty follows from facile hydrogen migrations in peptide radicals that can be induced by thermal collisional activation,13,14 so that standard product analysis by collision-induced dissociation (CID) cannot be used as a reliable structure probe. Therefore, new structure-specific © 2015 American Chemical Society
Received: October 21, 2015 Accepted: November 12, 2015 Published: November 12, 2015 4722
DOI: 10.1021/acs.jpclett.5b02354 J. Phys. Chem. Lett. 2015, 6, 4722−4727
Letter
The Journal of Physical Chemistry Letters
Scheme 1. Principal Dissociations of z4(•AWAR+) Ions Involving α and β Hydrogen Atom Migrations Induced by Collisional Activation (Blue-Coded) and UV Photodissociation (Red-Coded)
Photodissociation of z4(•AFAR+)(m/z 448) gave the action spectrum showing four distinct bands at 240, 275, 316, and 351 nm with a long-wavelength tail extending to 380 nm (Figure 2a). Most of the photofragment ion current was carried by [y3 − 2H]+ (m/z 391), [y2 − 2H]+ (m/z 244), and [x2 − H]+ (m/z 272), which are all even-electron ions that lack radical chromophores. The population homogeneity of the z4(•AFAR+) ions was probed by pulse-dependent measurements at 275, 316, and 351 nm. The 275 nm photodepletion curve (Figure S3, Supporting Information) showed an exponential decay, indicating complete dissociation of all ion species at this wavelength; however, the photodepletion curves measured at 316 and 351 nm were multimodal. The 351 nm curve was fitted with a composite function I(n)/I0 = 0.627e−0.0493n + 0.373 (rmsd = 0.027, Figure S4), indicating the presence of 37% of ion isomers not absorbing light at 351 nm. The 316 nm photodepletion curve also indicated ca. 40% of nonabsorbing species, although the descending part of the curve did not give a tight fit for a single exponential decay (Figure S5). TD-DFT Analysis of AWAR Radical Isomers. To interpret the UV action spectroscopy data, we carried out extensive TD-DFT calculations of excited-state energies and oscillator strengths of z4(•AWAR+) and z4(•AFAR+) isomers differing in the position of the radical defect. The TD-DFT methods used here were previously benchmarked on high-level equation-of-motion coupled-cluster excited-state calculations that identified the M06-2X,19 LC-BLYP,20 and ωB97X-D21 hybrid functionals as giving the closest fits of excitation energies for chromophores pertinent to peptide radicals.22 The z4(•AWAR+) radicals were considered as five valencebond isomers (1−5) differing in the position of the radical defect, each in multiple conformations. The lowest-energy conformers for each radical type are shown in Figure 1, and the others are in Figure S6. Ions 1a−1d are conformers having the radical defect at Cα of the truncated N-terminal Ala-2 residue (numbering as in the original AAWAR peptide). Conformers
ionization, stored in the linear ion trap (LIT), and dissociated by electron transfer in an ion−ion reaction with the fluoranthene anion. The z4 ions formed were selected by mass, stored in the LIT, and irradiated by 3 ns laser pulses at wavelengths that were varied from 210 through 400 nm. Photofragment ions were monitored at m/z values >220. Full experimental details are given in the Supporting Information. UV Action Spectra. Photodissociation of z4(•AWAR+) (m/z 487) was monitored for several product ion channels. The overall action spectrum, constructed from the sum of normalized fragment ion intensities, shows three major bands at 220, 264, and 358 nm (Figure 1a). The wavelength and mass-resolved relative intensities of the two most abundant photofragment ions, [y2 − 2H]+ (m/z 244) and [z4 − H]+ (m/ z 486), are plotted in Figure S1a,b (Supporting Information). The latter of these dominated in the
