w‐Type ions formed by electron transfer dissociation of Cys‐containing peptides investigated by infrared ion spectroscopy

Abstract In mass spectrometry‐based peptide sequencing, electron transfer dissociation (ETD) and electron capture dissociation (ECD) have become well‐established fragmentation methods complementary to collision‐induced dissociation. The dominant fragmentation pathways during ETD and ECD primarily involve the formation of c‐ and z •‐type ions by cleavage of the peptide backbone at the N─Cα bond, although neutral losses from amino acid side chains have also been observed. Residue‐specific neutral side chain losses provide useful information when conducting database searching and de novo sequencing. Here, we use a combination of infrared ion spectroscopy and quantum‐chemical calculations to assign the structures of two ETD‐generated w‐type fragment ions. These ions are spontaneously formed from ETD‐generated z •‐type fragments by neutral loss of 33 Da in peptides containing a cysteine residue. Analysis of the infrared ion spectra confirms that these z •‐ions expel a thiol radical (SH•) and that a vinyl C═C group is formed at the cleavage site. z •‐type fragments containing a Cys residue but not at the cleavage site do not spontaneously expel a thiol radical, but only upon additional collisional activation after ETD.


| INTRODUCTION
Mass spectrometry data in combination with bioinformatics algorithms form a key technology for peptide and protein sequencing, including the identification of posttranslational protein modifications. 1,2 Particularly with the advent of top-down proteomics and the sequencing of posttranslational protein modifications, electron induced dissociation methods-either electron capture or electron transfer dissociation (ECD or ETD)-have become popular. 3,4 The dominant ETD and ECD fragmentation pathways involve cleavage of the N-C α bonds resulting in the formation of c-and z • -type ions [5][6][7][8] (see Scheme 1). Neutral loss fragments including those from the residue side chains are also commonly observed. [9][10][11][12][13][14][15][16] Side chain losses can confirm sequence assignments, improve database matching scores, and can be useful in de novo sequencing 14 . It is therefore useful to train sequencing algorithms to recognize neutral loss fragments in addition to sequence ions. 17 Neutral losses from residue side chains in radical ExD sequence ions have for instance been used to distinguish between leucine and isoleucine residues and to identify isomeric amino acid combinations. [17][18][19][20] On the other hand, extensive formation of nonsequence ions due to entire or partial side-chain losses, especially when remote from the backbone cleavage site, may severely complicate database searching. Mechanistically, neutral loss from radical z • -type ions has been explained by several charge-remote fragmentation pathways. 10,[21][22][23] Migration of the radical may occur from the original site of cleavage at the C α atom to the C β and C γ positions of the same residue or to C α , C β , and C γ atoms in neighboring residues. 14,18,20,21,24,25 Subsequent expulsion of a neutral radical generates even-electron fragment ions in the mass spectrum, which have been suggested to adopt a w-type fragment ion structure by cleavage of the C β ─C γ bond of the N-terminal residue and the formation of a double bond between the C α and C β atoms (direct formation). Alternatively, cyclic u-type fragments may form as a result of bond formation between the N-terminal C α atom and the C β atom of the adjacent residue. 16,18,21,26 A possible mechanism for the loss of the entire side chain involves H-atom abstraction from the C γ and subsequent α-cleavage and expulsion of the side chain as an even-electron neutral species, leaving the radical on the peptide backbone. 21 Fung and Chan systematically investigated neutral loss product ions from ECD-generated z • -type ions using the doubly charged peptides [RGGGXGGGR+2H] 2+ , where X denotes one of the 20 naturally occurring amino acid residues. 27 Residues that were shown to undergo secondary loss of a neutral radical, thus leaving behind an even-electron charged fragment, include among several others cysteine (Cys), the subject of investigation here. Even-electron neutral losses from residue side chains were also reported for various other amino acid residues. 8,24,27,28 Cys-specific ExD pathways have been discussed in several studies. 23,27,29 For z • -type fragments from Cys-containing peptides, secondary fragmentation by loss of a 33-Da neutral fragment is common and is attributed to loss of a thiol radical (SH • ). For z 6and larger z • -type ions, neutral loss of CH 2 S (46 Da) has also been observed. 27 ETD on [AACAR+2H] 2+ generated the z 4 -ion (m/z 404) and the z 3 -ion (m/z 333), where the intensity of the z 3 -ion was significantly decreased by spontaneous loss of the SH radical, forming a secondary product at m/z 300. 29 ECD studies on peptides containing modified cysteine residues, such as carboxymethylated or carbamidomethylated Cys residues, report neutral losses corresponding to the modified Cys side chain. 30 The odd-electron neutral loss of C 2 H 4 NOS • (90 Da) has also been observed in a negative electron-transfer dissociation process on a doubly negatively charged ion containing carbamidomethylated Cys residues. 31 In related studies not actually involving ExD, the CID fragmentation behavior, and possible radical migration in open-shell cations of Cys and Cys-containing peptides have been studied by theoretical 31-33 as well as experimental methods. 16,23,[35][36][37][38][39][40] In experimental studies, CID-induced loss of nitric oxide from protonated S-nitrosocysteine is an efficient way to form radical cations of Cys and Cys-containing peptides. 35,36 Hao and Gross 36 showed that CID fragmentation of Cys-containing peptide radicals yields fragmentation similar to ExD experiments. Fragmentation reactions of radical cations of Cyscontaining di-and tripeptides were found to be radical-driven or charge directed, leading to losses of SH • and CH 2 S, respectively. 23 Analogous CID fragmentation products were found for [GlyCysArg] • + 38 . Observed CID products of the radical cation of the Cys amino acid were HOCO • , CH 2 S, CH 2 SH • , and H 2 S, 35 while Cys-containing peptide radicals expel SH • and CH 2 S. 35 Theoretical investigations suggested that loss of a HOCO • radical is energetically favored for the amino acid, followed by loss of the thiol radical and loss of the side chain (CH 2 SH • ). [32][33][34] Although the vinyl structure of the w-type fragment ion has often been assumed (see Scheme 2), experimental verification of this structure is scarce. Here, we use infrared multiple-photon dissociation (IRMPD) spectroscopy [41][42][43][44][45][46][47][48] to experimentally confirm their structure for two z-33 ETD fragments from Cys containing peptides. Several previous reports have made use of IR and UV photodissociation action spectroscopy methods to characterize ETD and ECD product ion structures. 41,47-57

| IRMPD spectroscopy
The peptides AAAACAK and AAACAAK were purchased from BIOMATIK, Canada, and used without further purification. Doubly protonated peptide ions were generated by electrospray ionization from 10 −7 -M solutions in 50:50 acetonitrile : water with 0.5% formic acid and mass selectively stored in a modified 3-D quadrupole ion trap mass spectrometer (Bruker, AmaZon Speed ETD). 42,48 The z •and w-type fragment ions were produced via an ion-ion reaction with the fluoranthene radical anion for 300 ms. Ions of interest were then mass isolated in the trap, and their IRMPD spectra were measured using the free electron laser FELIX. 58,59 Ions were irradiated by two macropulses, each pulse having an energy of 10 to 60 mJ, at a repetition rate of 10 Hz and a bandwidth of~0.5% of the center frequency. Relating parent and fragment ion intensities as the fragmentation yield (ΣI [fragment SCHEME 1 Overview of expected electron transfer dissociation sites cleaving the peptide backbone at one of the N─C α bonds, generating N-terminal c-ions and/or C-terminal z-ions. The z-ions are radical species SCHEME 2 z-Type electron transfer dissociation fragments expected for z-ions with a Cys residue at the cleavage site are not observed. Instead, a w-type ion lower in mass by 33 Da is observed due to additional neutral loss of a thiol radical. These w-type ions have generally been hypothesized to possess a vinyl group at the fragment's N-terminus ions]/ΣI [parent + fragment ions]), and plotting the yield as a function of laser frequency generates an infrared spectrum. The yield is linearly corrected for frequency-dependent variations in the laser power, and the infrared frequency is calibrated using a grating spectrometer.

| Computational chemistry
Optimized molecular geometries and theoretical infrared spectra were obtained using density functional theory (DFT) calculations as implemented in Gaussian 09 revision D01. 60 The potential energy surface was explored to locate the lowest energy minima by using a molecular mechanics/molecular dynamics approach employing AMBER 12. 61 Within AMBER an initial geometry optimization has been performed, followed by a simulated annealing procedure up to 500 K yielding  Figure 1 shows the ETD mass spectra for the two doubly protonated heptapeptides AAAACAK and AAACAAK investigated in this study, having a Cys residue in the third and fourth position from the C-terminus, respectively. The identified ETD fragment ions are annotated in Figure 1. As a consequence of the basic Lys residue at the C-terminus, the series of z • -type fragment ions are nearly complete, except for the smallest ions.

| RESULTS AND DISCUSSION
Interestingly, for AAAACAK-with Cys in the third position from the C-terminus-the z 3 fragment is missing, but instead, a peak 33 Da lower in mass is observed. Analogously, for AAACAAK, the z 4 ion is missing and instead a peak at the nominal mass of z 4 -33 is observed. We observe that neutral loss of a thiol radical (SH • ) occurs only for z • -ions that contain a cysteine residue at the cleavage site; these fragments are commonly referred to as w-type ions.  The experimental spectra are compared with predicted spectra for the lowest energy structures (red) and to the qualitatively best matching calculated spectra (blue). Relative energies (kJ/mol) at the B3LYP, M06-2X, and MP2(full) levels of theory are given in the square brackets to involve a double bond between the αand β-carbon atoms of the residue at the cleavage site. This fragment ion structure was hypothesized on the basis of differences in the CID MS/MS behavior of z •ions versus (z + 1)-ions. 20 Related, the differences in bond dissociation energies for H-atom removal from the α and β carbons have been used to rationalize the CC double bond structure of the w-ion. 16,21 Computational investigations of the potential energy surface of the dissociation reaction at the DFT level also indicate the formation of a vinyl group at the N-terminus of the w-ion. 27 However, direct experimental probing of the w-ion structure has until now not been reported to our knowledge. In this contribution, IR spectroscopy of two w-type ions formed by loss of an SH-radical from a Cys residue at the cleavage site confirms the vinyl group structure suggested previously. 14,16,18,21,27,31 Indirectly, the observation of this structure also confirms the suggested charge remote fragmentation pathways suggested. [21][22][23]27,31 Other z • -type ions were observed to expel a thiol radical only upon applying additional CID activation after ETD, in line with reported higher rates of formation of w-ions compared with u-ions. 18 Also in accordance with previous studies, 27,29 loss of CH 2 S (46 Da) after collisional activation was only observed for z • -ions that contain a Cys residue, but not at the cleavage site. Spectroscopic probing of the structure of these fragment ions is challenging because of the more involved ETD/CID MS n scheme required to produce them, and the inherent lower ion yields as compared with the w-type ions; such experiments are reserved for future studies.

| CONCLUSION
We have reported the first infrared spectra for w-type fragment ions resulting from ETD of two Cys-containing peptides. Specifically, the mass peak at m/z 272 in the ETD mass spectrum of [AAAACAK +2H] 2+ and that at m/z 343 in the ETD mass spectrum of [AAAC AAK+2H] 2+ , which cannot be assigned to sequence ions but are the result of neutral SH • -loss from the radical z 3 and z 4 -ions of the respective peptides, have been investigated with infrared ion spectroscopy. Evaluation of experimental and predicted IR spectra confirm that these w-type fragment ions possess a structure having a C═C double bond at their N-terminus. This structure supports the mechanism proposed for the formation of w-type fragment ions in ExD, proceeding through cleavage of the C β -S bond of the Cys residue with concomitant formation of a double bond between the C α and C β atoms of the w-ion. Elimination of the SH • -group is only observed for z • -ions with a cysteine residue at the cleavage site, independent of its position of cysteine in the peptide.