# Unravelling the structure of one-electron oxidation products of model peptide backbones containing methionine by IRMPD spectroscopy: the effect of the neighbouring groups

Yining Jiang, Carine Clavaguera, Marc Lecouvey, Chantal Houée-Levin,

Jonathan Martens, Giel Berden, Jos Oomens, Debora Scuderi

► To cite this version:

Yining Jiang, Carine Clavaguera, Marc Lecouvey, Chantal Houée-Levin, Jonathan Martens, et al. Unravelling the structure of one-electron oxidation products of model peptide backbones containing methionine by IRMPD spectroscopy: the effect of the neighbouring groups. ChemPhysChem, 2024, 10.1002/cphc.202400918. [hal-04785453](https://hal.science/hal-04785453v1)

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# Unravelling the Structure of One-Electron Oxidation Products of Model Peptide Backbones Containing Methionine by IRMPD Spectroscopy: The Effect of the Neighbouring Groups

Yining Jiang,[a] Carine Clavaguéra,[a] Marc Lecouvey,[b] Chantal Houée-Levin,[a] Jonathan Martens,[c] Giel Berden,[c] Jos Oomens,[c, d] and Debora Scuderi*[a]

A series of Methionine (Met) derivatives, where either the amino group and/or the carboxylic acid group is blocked by acetyl and/or methyl ester functionalities, has been investigated by Collision Induced Dissociation-tandem mass spectrometry (CID-MS²) and Infrared Multiple Photon Dissociation (IRMPD) spectroscopy. The CID-MS² experiments were performed using a Fourier-transform ion-cyclotron-resonance (FT-ICR) mass spectrometer equipped with an electrospray ionization (ESI) source. The IRMPD spectra were recorded employing a Paul type ion-trap coupled with the free-electron laser (FEL) FELIX in the

fingerprint region from 600 to 2000 cm⁻¹. We show that the oxidation of the methionine residue with protected terminal groups induces the formation of a sulfoxide function. However, compared to the IRMPD spectrum of protonated methionine and methionine sulfoxide, significant spectral differences are observed in the spectra of model protected peptide backbones containing methionine. DFT calculations show that protonation occurs on the sulfoxide group in the gas phase for these model peptide backbones containing methionine, shifting the diagnostic signature of the sulfoxide group from 1000 to 862 cm⁻¹.

## Introduction

Methionine (Met) is an essential amino acid and its thioether function is one of the main targets of oxidizing free radicals. In proteins, the main final compound is most often methionine sulfoxide (MetSO).[1] MetSO can be repaired by methionine sulfoxide reductases, so that this amino acid residue could act as a molecular switch to detoxify oxidants and protect important regions of the protein. However, when it is not repaired or when the final product of oxidation is not MetSO, its oxidation is a highly damaging event that has been associated with pathogenesis and the aging process.[2] In one-electron oxidation of Met, hydroxyl radicals attack the sulphur atom to

form an S-centered radical cation (>$S^{•+}$). This radical cation forms a 2-center-3-electron (2c–3e) bond with any atom having a lone pair of electrons. The mechanism of the one-electron oxidation of Met varies with the environment of the thioether function and by consequence the yield of sulfoxide, formed during oxidation, may depend on several yet unidentified factors, among which the geometry of the compound.[3] Stabilization of sulphur radical cations in peptides derives mainly from the lone pair of electrons on the C- and N-terminal moieties of the peptides. However, it has been confirmed in transient species that the sulphur radical cations can also been stabilized by bonding to the heteroatoms involved in peptide bonds.[4] The types of 2c–3e intermediates depend on the structural, geometrical, and conformational properties of peptide molecules, such as (1) the location of the Met residue in the peptide, (2) the protection of N/C-terminal amino/carboxylate groups through N-acetylation or amidation/esterification, (3) the nature of the peptides (open/cyclic), (4) the number of Met residues, and (5) the configuration (L/D) of the Met residues. All these geometrical and structural properties may have an effect on the final products of oxidation.

In previous experiments, we investigated by tandem mass spectrometry and IRMPD spectroscopy the effect of an adjacent Met residue on the final products of one electron-oxidation using Met-Met dipeptides (linear and cyclic) as models.[5,6] Peptides containing multiple Met residues in their sequence are good models for proteins. The side chains in the naturally occurring linear peptides and proteins are forced to be spatially in opposite direction due to the formation of trans-amides in the peptide bond. However, a change in the configuration from L–L to L–D leads to an opportunity to study the effect of the interactions between two adjacent side chains. Cyclic peptides

[a] Y. Jiang, C. Clavaguéra, C. Houée-Levin, D. Scuderi
Université Paris-Saclay, CNRS, Institut de Chimie Physique, UMR8000, 91405 Orsay, France
E-mail: debora.scuderi@universite-paris-saclay.fr

[b] M. Lecouvey
Département of Chemistry, Université Sorbonne Paris Nord, CNRS, Laboratoire de Chimie Biorganique Biophysique et Biométrieau, UMR 7244, 1 rue de Chablis, Bobigny, F-93000, France

[c] J. Martens, G. Berden, J. Oomens
Radboud University, Institute for Molecules and Materials, FELIX Laboratory, Toernooiveld 7, Nijmegen, 6525 ED, Netherlands

[d] J. Oomens
Van't Hoff Institute for Molecular Sciences, University of Amsterdam, P.O. Box 94157, Amsterdam, 1090 GD, Netherlands

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Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cphc.202400918

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© 2024 The Authors. ChemPhysChem published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
are in this context an interesting class of molecules as models for the interior of (linear) proteins, where terminal groups are absent. We were able to show that the final products of oxidation are the same regardless of the structure of the precursor sulphur centered free radical. We have also investigated designed peptides with variable-length proline bridges.[7] We selected Met-(Pro)n-Met peptides, where Met residues are separated by n Pro residues, with n = 0, 1, 2. The use of such peptides allows for distance control between the sulphur atoms in the Met side chains. This approach helped to study the effect of long-range electron transfer (ET) between amino acid residues on the final products formed in the oxidation process. The effect of possible intramolecular contact formation in the stabilisation of the sulfur-centred radical cation during γ radiolysis in γ-Glu-(Pro)n-Met has been investigated as well, where the N-terminal Met is replaced by γ-Glu.[7] In the case of Met-(Pro)n-Met, although the distance between two Met residues controls the nature of free radicals formed in the peptides, we showed that it has no significant effect on the final products of oxidation. These results confirmed that the final products are not controlled by free radical structure.

The aim of the present study is to shed light on the effect of N/C-terminal amino/carboxylate groups through N-acetylation or amidation/esterification on the nature and structure of final products of oxidation of model peptides containing methionine by IRMPD spectroscopy. The acetylation of the N-terminal amino group eliminates the fast-intramolecular proton transfer from the amino group to the sulfuranyl moiety, which was suggested earlier as the main decay reaction pathway, while the esterification of the C-terminal carboxyl group eliminates its decarboxylation.[8] Small methionine derivatives as well as a model-designed peptide containing methionine are investigated by mass spectrometry and IRMPD spectroscopy in order to correlate the structural modifications in the designed model peptides with the final products observed.

The terminal protected methionine derivates under study are shown in Figure 1. In particular we investigated N-terminal acetyl protected Met (NAcMet), Met with N- and C-terminals protected by acetyl and methyl ester groups, respectively (NAcMetOCH3), and Met-Met dipeptide with terminals protected by acetyl and methyl ester groups (NAcMetMetOCH3). NAcMetSO is a commercial product with a known structure, which is employed as a reference to be compared with the oxidation product of NAcMet.

By comparing the experimental results obtained by IRMPD spectroscopy for different terminal-protected Met with unprotected Met, we elucidate the influence of terminal protecting groups on the spectroscopic features observed in the IR spectra of the oxidation products. These spectroscopic features can be used as diagnostic signatures for the identifications of the final oxidation products. The MetMet dipeptide with both sides protected is also studied, providing information on the interaction between the sulphur centers from neighbouring side chains, without interference by two terminal groups.

## Materials and Methods

### Materials

NAcMet was purchased from Aldrich-Sigma and NAcMetOCH3 from NovoPep. NAcMetSO was purchased from CHEM-IMPEX. NAcMet-MetOCH3 was synthesized in the group of Prof. Marc Lecouvey at Sorbonne University Paris Nord. All chemical products were used as received without further purification. The linear peptide NAcMet-MetOCH3 was prepared in 2 steps. The synthesis started by the protection H-Met OH amino group. The aminoacid H-Met OH was refluxed by a mixture of acetic anhydride in acetic acid for 1 h 30. After evaporation, N-acyl amino methionine was obtained in the form of white powder in 40% yield. The coupling reaction with (L) H-Met-OMe was carried out by using hydroxybenzotriazole and EDC-HCl as activating reagents. After one night of reaction, the mixture is extracted with AcOEt and the organic layer was evaporated to get a white powder. The dipeptide was purified after recrystallization in a mixture of petroleum ether and ethyl acetate in 58% yield.

### Gamma Source (γ) Radiolysis

Samples were dissolved in ultra-pure water at a concentration of 10⁻³ M for γ-irradiation. Prior to irradiation, samples were purged by bubbling with N₂O (nitrous oxide with a purity of 99.998% was delivered by ALPHA GAZ) for 30 min to increase the final *OH yield and suppress the highly reductant e⁻aq according to the reaction

$$e^{-}_{aq} + N_{2}O + H_{2}O \rightarrow *OH + OH^{-} + N_{2}$$

The rate constant of this reaction has been determined to be (9.1 ± 0.2) × 10⁹ M⁻¹s⁻¹.[9,10] which almost doubles the yield of *OH compared to conditions without N₂O. In order to initiate hydroxyl-induced oxidation, γ-irradiation was carried out by using the panoramic ⁶⁰Co γ-source IL60PL Cis-Bio International (France) at Paris-Saclay University (Institute of Physical Chemistry, Orsay, France). The dose rate of the gamma source was determined by

<figure><img src="image_5.png" /><figcaption>Figure 1. Chemical structures of protected methionine. (a) NAcMet (MW = 191 g/mol), (b) NAcMetSO (MW = 207 g/mol), (c) NAcMetOCH3 (MW = 205 g/mol) and (d) NAcMetMetOCH3 (MW = 336 g/mol).</figcaption></figure>
Fricke dosimetry.[11] Samples were irradiated with a dose of 833 Gy. The dose rate was equal to 25.45 Gy.min⁻¹ by Fricke dosimetry and then corrected by the decay of the source over time. All irradiations were performed at room temperature. In the irradiation process, the relevant reaction is[12]

$$H_2O \rightarrow (0.28)^*OH, (0.062)^*H, (0.28)e^{-aq}, \\ (0.047)H_2, (0.073)H_2O_2, (0.28)H_3O^+$$

The chemical yields of each species after radiolysis are given in brackets in µM/J (known as G-values).

## FT-ICR MS²-CID Analysis

After irradiation, the freshly irradiated samples were diluted in 50/50 water/methanol containing 0.1% formic acid to a concentration of 50 µM for MS-based analysis. Non-irradiated solutions (with the same molecules dissolved in water and purged by N₂O) at the same concentration was set as reference. A 7 T hybrid FT-ICR mass spectrometer (APEX-Qe Bruker), in Orsay, France, has been used to identify all species generated in the irradiated solution.[13,14]

The peptides were delivered into gas-phase by an electrospray ionization (ESI) source with a flow rate of 120 µL h⁻¹ and they were investigated in positive ion mode. Typical extraction voltage was -4500 V and the desolvation temperature was 200 °C. The dry gas flow was fixed at 8 L min⁻¹ and the nebulizer pressure was set to 1.5 bar. Ions were accumulated, transferred through ion transfer optics, thermalized in a quadrupole-hexapole (Qh) interface and later extracted out towards the FT-ICR cell, which is maintained under high vacuum (< 10⁻⁹ mbar) at room temperature. Mass spectra of the species were then recorded with an appropriate accumulation time depending on the abundance of species of interest, typically 1 s.

For processing the collision induced dissociation (MS²-CID) mass spectra, target ions were firstly selected using an isolation window of 2 Da by a resonant radiofrequency (RF) pulse in FT-ICR mass spectrometer. MS²-CID was carried out by introducing Argon buffer gas into the collision chamber, raising the energy of the ions by kinetic-to-internal energy conversion and leading to fragmentation. Fragment ions were then pulse-extracted and detected in the ICR cell.

## IRMPD Spectroscopy

IR multiple-photon dissociation (IRMPD) is a process that involves the successive absorption of multiple photons and the dispersion of energy in the molecule by intramolecular vibrational redistribution (IVR).[15] At absorption wavelengths of the (mass-selected) ion, the internal energy reaches and exceeds the fragmentation threshold and fragment ions are observed in the mass spectrum recorded.[16,17] In this work, the IRMPD spectroscopy experiments have been carried out at the Free Electron Laser For Infrared Experiments (FELIX) Laboratory in Nijmegen (Netherlands).

The IRMPD experiments are performed on a Bruker AmaZon quadrupole ion trap mass spectrometer[18] coupled with the free-electron laser FELIX.[19] The AmaZon ion trap is equipped with an ESI source. Ions are infused using a syringe pump at a rate of 120 µL/h, assisted by a pressurized nebulizing N₂ gas heated to 200 °C. The spray voltage is 3–5 kV. The ring electrode of the trap has been modified by drilling two Ø3 mm holes at the top and bottom, allowing the IR beam to enter the trap, interact with ion cloud and conduct laser spectroscopy.

The FELIX IR-FEL runs at 10 Hz pulse repetition rate and covers wavelengths over the entire fingerprint region. Macropulses of 5–10 µs consist of a train of ~5 ps micropulses, spaced by 1 ns. The energy of each macropulse reaches up to ~150 mJ, while the spectral width is ~0.5% of the central wavelength (λc = 0.5%). Five neutral-density IR attenuators (of 3, 5, 8, 10 and 10 dB used in adjustable combinations) provide attenuation levels to adapt the pulse energy to the studied systems,[20] such as to prevent saturation of the IRMPD signal resulting from full depletion of the precursor ion population. A laser energy meter is placed at the exit of the optical pathway from AmaZon mass spectrometer to record the pulse energy after passing through the ion trap. The IRMPD spectra were obtained by plotting -ln(P/P₀) as a function of the IR wavenumber, where P represents the intensity of precursor ion mass peaks and F the summed intensity of IR induced fragment ions. The spectral intensity was multiplied by attenuation factor selected and linearly corrected for fluctuations in the laser power over the frequency scan range. The frequency was calibrated with a grating spectrometer with an accuracy of ±0.02 µm.

## DFT Calculations

Calculations have been carried out for NAcMetH⁺, NAcMetSOH⁺, [NAcMet-H⁺]⁻ and [NAcMet(O)-H⁺]⁻. Conformations have been explored extensively. The iterative metadynamic sampling and genetic crossover (iMTD-GC) method implemented in the Conformer Rotamer Ensemble Sampling Tool (CREST) program[21,22] have been used for the conformer sampling. The sampling included additional geometry optimization of the final conformer ensemble. The calculations of the conformer energies are based on the semi-empirical extended tight-binding method (GFN2-xTB).[23] The possible tautomeric conformers have been examined as well. Geometry optimizations, IR spectral simulations and energy calculations were carried out with the Gaussian 16 package of programs.[24]

The GFN2-xTB structures were then optimized at the B3LYP/6-311+G(d, p) level of theory and the harmonic IR absorption spectra were computed. A frequency scaling factor 0.98 has been applied for a better agreement between the theoretical spectra and experimental ones in the fingerprint region. For all the molecules, the frequencies associated with sulfoxide group were left unscaled, which complies previous evidence.[25-28] The calculated frequencies were convoluted with a 20 cm⁻¹ full width at half maximum (FWHM) in Lorentzian line shape over the fingerprint region for all molecules. Spectral assignments have been achieved by comparing the experimental IRMPD spectra with the calculated IR spectra.

# Results and Discussion

## MS and CID-MS²

Mass spectra of the irradiated and non-irradiated NAcMet, NAcMetOCH₃ and NAcMetMetOCH₃ solutions are shown in Figure 2a, b and c, respectively. By comparing the mass spectra of non-irradiated and irradiated solutions we can observe that NAcMet shows, after γ-radiation, the dehydrogenation (−2 Da) and the singly oxidized product (+16 Da). NAcMetOCH₃, oxidation induces the dehydrogenation (−2 Da) as well, singly oxidized (+16 Da) and doubly oxidized (+32 Da) products. In the case of NAcMetMetOCH₃ singly (+16 Da) and doubly (+32 Da) oxidized products are visible, after radiolysis-induced oxidation.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2.** Mass spectra of non-irradiated (black) and irradiated (red) (a) NAcMet (b) NAcMetOCH₃ (c) NAcMetMetOCH₃ (irradiation dose 833 Gy).</figcaption></figure>

We previously showed that the product of dehydrogenation originates from the loss of H₂ from the Met residue.[29] No or very little decarboxylation products (–44 Da) have been observed after irradiation in this case, even though there is a no-protected C-terminal in NAcMet. In the process of *OH-induced Met oxidation, the decarboxylation is accompanied by the formation of *α*-N radicals via a transfer of the oxidation site from sulphur to the amino group.[30] By time-resolved pulse studies, terminal-protected Met residues have been found to display very low yields of the reducing radicals at short time scales.[31,32] In our experiments, the protection of terminal groups hampers the loss of CO₂ and the formation of *α*-amino radicals in species with a single Met residue and we observe that the final yield of decarboxylation products decreases due to the protection of terminal groups at long time scales.

CID-MS² experiments have been carried out for NAcMetH⁺, [NAcMet-H⁺]⁻, NAcMetOCH₃H⁺ and NAcMetMetOCH₃H⁺ and their oxygen-addition products, see Figure S1-4. The most intense CID-MS² fragments are listed in Table S1-4 with tentative annotations.

NAcMet(O)H⁺ shows the same fragmentation channels in CID-MS² compared to the commercial product NAcMetSOH⁺, suggesting that they possess the same molecular structure. The common fragment at *m/z* 144, corresponding to the loss of HSOCH₃, for protonated NAcMet and NAcMet(O) suggests that the addition of one O-atom in NAcMet(O) occurs on the side chain of Met residue and hence forms a sulfoxide group.

NAcMet and its oxo-form at +16 Da were investigated in negative ion mode as well. The CID-MS² fragmentation mass spectra are shown in Figure S12. Tentative assignments of the observed fragments are listed in Table S12. The fragmentation mass spectra of [NAcMet-H⁺]⁻ and [NAcMet(O)-H⁺]⁻ present two common dissociation channels: the loss of O=C=CH₂ (–42 Da) and of the Met side chain (–48 Da for non-oxidized; –64 Da for oxidized). The fragment at *m/z* 142 indicates that the addition of one oxygen atom occurs on the S-containing Met side chain forming a sulfoxide group.

CID-MS² fragmentation mass spectra of NAcMetOCH₃H⁺ and its singly oxidized form, are shown in Figure S13 with tentative fragment assignments in Table S13. The fragmentation mass spectrum of the precursor NAcMetOCH₃H⁺ gives three main fragments at *m/z* 174, *m/z* 158 and *m/z* 164. The fragmentation of the +16-mass increment product, NAcMetO(O)CH₃H⁺ (*m/z* 222), yields two fragments at *m/z* 190 and *m/z* 158, respectively. Based on the presence of a common fragment at *m/z* 158 for both non-oxidized and singly oxidized species, we infer that the additional O-atom of [NAcMetOCH₃ + 16] is localized on the S-containing side chain and hence that oxidation leads to the formation a sulfoxide group.

The CID-MS² results of protonated NAcMetMetOCH₃ and its oxo-forms +16 and +32 are shown in Figure S14 with corresponding fragment assignments in Table S14. In the fragmentation patterns of singly and doubly oxidized products, NAcMetMet(O)OCH₃H⁺ and NAcMetMet(O)₂OCH₃H⁺, the *b*₁ and
<figure><img src="image_8.png" /></figure>

y₁ fragments with the addition of one oxygen atom are observed, respectively. Therefore, it can be concluded that the oxygen atoms were added on the Met side chain of both N- and C-terminal residues. Diagnostic loss of HSOCH₃ was observed in the fragmentation of NAcMetMet(O)CH₃H⁺, while the loss of HSOCH₃ and HSO₂CH₃ were observed in the fragmentation pattern of NAcMetMet(O)₂CH₃H⁺. This indicates that the single oxidation product NAcMetMet(O)OCH₃ is composed of sulfoxide formation in one of the two Met residues, while the double oxidation product NAcMetMet(O)₂CH₃ is a mixture of sulfoxide formation in both Met residues and sulfone function formation in one of the two Met residues.

## IRMPD Spectroscopy Results

IRMPD spectroscopy experiments have been carried out in both positive and negative mode for NAcMet. The IRMPD spectra of NAcMetH⁺ (m/z 192), NAcMet(O)H⁺ (m/z 208) and NAcMetSOH⁺ (m/z 208) have been recorded from 600 to 1900 cm⁻¹, see Figure 3.

The IRMPD spectra of NacMet(O)H⁺ and of the reference sample NacMetSOH⁺ have the same spectral features, indicating that they have the same chemical structure as the additional O-atom in the NAcMetSO reference compound resides on the sulphur atom of the Met residue. Major spectral differences between the non-oxidized and oxidized species are observed below 1000 cm⁻¹. NAcMet(O)H⁺ and NAcMetSOH⁺ show an intense band centered at 862 cm⁻¹ with a blue-side shoulder at 975 cm⁻¹, whereas NAcMetH⁺ possesses a sharp band at 741 cm⁻¹. In the 1500–1800 cm⁻¹ energy region, NAcMetH⁺ exhibits two extra absorption bands at 1612 and 1705 cm⁻¹, respectively. The differences in the 750–900 cm⁻¹

energy region are due to oxidation-induced structural modifications in the oxidized molecule.

MetH⁺ and MetSOH⁺ IRMPD spectra have already been reported.[33] We will discuss below the IRMPD spectra of NAcMetH⁺ and NAcMet(O)H⁺ aiming to unravel the spectral difference induced by protection of the terminal groups in Met. For the sake of clarity, a comparison of the IR spectra of MetH⁺ and MetSOH⁺ with NAcMetH⁺ and NAcMet(O)H⁺ respectively are reported in Figure SI5.

As expected, the IRMPD spectrum of NAcMetH⁺ is more complex⁺. Both bands are slightly red-shifted compared to the bands of MetH⁺. Protecting the N-terminal group by an acetyl group introduces an additional carbonyl group, which makes the fingerprint region of the spectrum more congested. The IRMPD spectra of NAcMet(O)H⁺ and MetSOH⁺ are identical, suggesting that the oxidation in NAcMet(O)H⁺ occurs on the sulphur atom. IRMPD spectroscopy experiments in negative mode have been performed as well for these systems. The comparison of the IRMPD spectra of [NAcMet-H⁺]⁻, [NAcMet(O)-H⁺]⁻ and the reference [NAcMetSO-H⁺]⁻ is reported in Figure 4. The IRMPD spectrum of [NAcMetO-H⁺]⁻, and that of [NAcMetSO-H⁺]⁻ are again highly overlapping. Comparing to the deprotonated non-oxidized molecule, [NAcMet-H⁺]⁻, the only significant difference is the appearance of a prominent band at 1054 cm⁻¹ in the spectrum of the oxidized species, corresponding to the sulfoxide stretching mode.

The IRMPD spectra of oxidized blocked Met products in positive and negative modes show a diagnostic S=O band around 1000 cm⁻¹; it is red shifted in positive mode compared to the negative one. We conclude that the sulfoxide group is probably protonated in the case of NAcMet(O)H⁺ and involved in a hydrogen-bond interaction.

The IRMPD spectra for NAcMetOCH₃ and for NAcMet(O)OCH₃ in positive mode are shown in Figure 5. The

<figure><img src="image_9.png" /><figcaption>**Figure 3.** Comparison of the IRMPD spectra of non-oxidized NAcMetH⁺ (black), singly oxidized NAcMet(O)H⁺ (red) and reference NAcMetSOH⁺ (green).</figcaption></figure>
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 4.** Comparison between the IRMPD spectra of non-oxidized NAcMetH⁺ (black), singly oxidized product [(NAcMet(O))-H⁺]⁻ (red) and reference [NAcMetSO-H⁺]⁻ (green).</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 5.** Comparison between the IRMPD spectra of non-oxidized NAcMetOCH₃H⁺ (black) and singly oxidized product NAcMet(O)OCH₃H⁺ (red).</figcaption></figure>

spectra of protonated oxidized and non-oxidized species present several spectral differences. An intense band at 862 cm⁻¹ and a medium band at 975 cm⁻¹ are observed for the oxidized form. Only a minor band at 948 cm⁻¹ is detected for the non-oxidized species, together with two less well resolved bands at 1619 and 1679 cm⁻¹. Similar spectral differences have been observed for NAcMet. The intense band at 862 cm⁻¹ can be assigned to the of S=OH⁺ vibration, based on the results for NAcMet(O)H⁺.

The IRMPD spectra of NAcMetOCH₃H⁺ and NAcMet(O)OCH₃H are discussed below on the basis of the IRMPD spectra of MetH⁺ and MetSOH⁺³³ to obtain an insight into the consequences of the absence of terminal groups in hydroxyl-induced oxidation of single Met.

The IRMPD spectrum of NAcMetOCH₃H⁺, with N- and C- terminals protected by an acetyl and a methyl ester group, respectively, shows significant changes if compared to the spectrum of protonated Met and MetSO. These spectral differ-
<figure><img src="image_13.png" /></figure>

ences are even more evident when we compare the spectrum of NAcMetH⁺ and NAcMetOCH₃H⁺. As expected, two terminal protecting groups induce a large number of absorptions and make the IRMPD spectrum congested. Some differences have been observed in the position of the S=O (~862 cm⁻¹) and S=OH⁺ (~969 cm⁻¹) bands, making them valuable diagnostic signatures of oxidation in Met.

The experimental IRMPD spectra of NAcMetMetOCH₃H⁺ (m/z 337), NAcMetMet(O)OCH₃H⁺ (m/z 353) and NAcMetMet(O)₂OCH₃H⁺ (m/z 369) in the fingerprint region of 600–1900 cm⁻¹ are reported in Figure 6. According to the CID-MS² results (Table S14), the +16-oxidation product corresponds to a mixture of sulfoxide formation on either of the two Met residues. The +32-oxidation product is assigned to a mixture of sulfoxide formation on both Met residues and sulfone group formation on one of the two Met residues. The spectra of NAcMetMetOCH₃H⁺ and those of its oxo-forms are congested and quite similar, as seen in Figure 6.

## DFT Calculations

A comparison of the calculated IR spectra of the most stable structures of NAcMetH⁺ with its experimental IRMPD spectrum is displayed in Figure 7. **acMH+_-1** is the most stable structure. The calculated IR bands of **acMH+_-1** can justify most of the bands observed in experimental IRMPD spectrum except the medium-intensity band L and the less-resolved band F. **acMH+_-2** presents a similar spectrum as **acMH+_-1** and it is only 2.1 kJ/mol higher in energy. Bands L and F require the presence of geometry **acMH+_-4** to be justified. In order to explain the presence of the shoulder L' in the experimental spectrum, it is necessary to admit the presence of **acMH+_-3**. **acMH+_-3** and

**acMH+_-4** lie 10.7 and 13.3 kJ/mol higher in energy than the most stable geometry **acMH+_-1**, respectively. Therefore, the experiments suggest in this case that **acMH+_-1**, **acMH+_-2**, **acMH+_-3** and **acMH+_-4** are populated under our experimental conditions. They are all protonated on the N-acetyl oxygen s. **acMH+_-1** and **acMH+_-2** are stabilized by two non-covalent interactions: C=OH⁺...S and NH...O=C. **acMH+_-3** is stabilized by the intramolecular interactions NH...S and C=OH+...O=C. **acMH+_-4** has a hydrogen bond interaction between the sulphur atom and the amide proton. Other explored structures with corresponding spectra are detailed in Figure S16-7.

The bands observed in the IRMPD spectrum of NAcMetH⁺ are listed and tentatively assigned in Table 1.

Calculations have also been carried out for the protonated oxo-form, NacMetOH⁺. Four representative structures at relatively low energy with their calculated spectra are provided in Figure 8 and compared with the experimental IRMPD spectrum.

**acMOH+_-1** is the most stable structure of NacMetOH⁺, although its calculated IR spectrum cannot justify all the features observed in the IRMPD spectrum of NacMetOH⁺. In particular **acMOH+_-1** shows a medium-intensity band centered at 1039 cm⁻¹ where no intense band is observed in the experimental spectrum. The IR calculated spectra of both **acMOH+_-2** and **acMOH+_-3**, higher in energy by 2.5 and 4.9 kJ/mol, respectively, show a better agreement with the IRMPD spectrum and therefore are likely populated under our experimental conditions. The protonation sites of **acMOH+_-2** and **acMOH+_-3** are on the acetyl oxygen and sulfoxide oxygen, respectively. **acMOH+_-2** is stabilized by a C=OH⁺...O=S hydrogen bond and an NH...O=C interaction. Similarly, **acMOH+_-3** is stabilized by an S=OH+...O=C hydrogen bond and an NH...O=C interaction. Other calculated structures at

<figure><img src="image_14.png" /><figcaption>**Figure 6.** Comparison between the IRMPD spectra of non-oxidized NAcMetMetOCH₃H⁺ (black), singly oxidized product NAcMetMet(O)OCH₃H⁺ (blue), and doubly oxidized product NAcMetMet(O)₂OCH₃H⁺.</figcaption></figure>
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>**Figure 7.** Comparison of the experimental IRMPD spectrum of NAcMetH⁺ with convoluted calculated IR absorption spectra of representative conformers of the lowest-energy geometries, optimized at the B3LYP/6-311++g(d,p) level of theory. The H-bond distances are reported in Å. The corresponding structures are reported on the right together with the relative Gibbs free energy (ΔG, in parentheses) at 298 K in kJ/mol, calculated at B3LYP/6-311++g(d,p) level of theory.</figcaption></figure>

higher energy with corresponding spectra are reported in Figure SI8–9.

A tentative assignment of the bands observed in the experimental spectrum of NacMetOH⁺ is reported in Table 2. Band B centered at 862 cm⁻¹ is assigned to the stretching of protonated sulfoxide group S=OH⁺ coupled with CC stretches. It is diagnostic for the formation of the oxo-form. Interestingly,

the spectral signature of sulfoxide group in the protonated oxo-
form is red-shifted from 947 cm⁻¹ in the case of MetSOH⁺³³ to
862 cm⁻¹ in the case of NacMetOH⁺ (Figure SI5). This is due to
the change in protonation site from the amino to the sulfoxide
group, induced by the N-terminal blockage. A similar behaviour
has been observed for cyclic dipeptides, where the protonation
site shifts from the amino nitrogen to the sulfoxide oxygen⁶.
<figure><img src="image_17.png" /></figure>

<table><caption>**Table 1.** Experimental and calculated vibrational frequencies for the selected isomers for NAcMetH⁺ at B3LYP/6-311+g(d,p) level of the theory, and corresponding band assignments.</caption><thead><tr><th></th><th>Experiment</th><th>acMH + _1</th><th>acMH + _2</th><th>acMH + _3</th><th>acMH + _4</th><th>Assignments</th></tr></thead><tbody><tr><td>A</td><td>741</td><td>754</td><td>754</td><td>747</td><td>722</td><td>vCC, δCNH, δCOH</td></tr><tr><td>B</td><td>927</td><td>931</td><td>921</td><td>909</td><td>929</td><td>vCC, δCOH, δCH<sub>3</sub></td></tr><tr><td>C</td><td>1148</td><td>1156</td><td>1159</td><td>1165</td><td>1143</td><td>vCO, δCOH, δCH<sub>2</sub></td></tr><tr><td>D</td><td>1227</td><td>1245</td><td>1241</td><td>1192</td><td>1204</td><td>vC=OH<sup>+</sup> (acetyl), δCOH, δCNH, δCH<sub>2</sub></td></tr><tr><td>E</td><td>1281</td><td>1278</td><td>1269</td><td>1289</td><td>1295</td><td>vCC, vCN, δCOH, δCNH, δCH<sub>2</sub></td></tr><tr><td>F</td><td>1318</td><td></td><td></td><td>1321</td><td></td><td>vCC, δCOH</td></tr><tr><td>G</td><td>1346</td><td>1353</td><td>1346</td><td></td><td>1357</td><td>vCC, δCOH, δCNH, δCH<sub>2</sub>, δCH<sub>3</sub></td></tr><tr><td>H</td><td>1400</td><td>1398</td><td>1401</td><td>1388</td><td>1391</td><td>vCC, δCOH, δCNH</td></tr><tr><td>I</td><td>1431</td><td>1447</td><td>1448</td><td>1444</td><td>1441</td><td>δCH<sub>2</sub>, δCH<sub>3</sub></td></tr><tr><td>J</td><td>1548</td><td>1553</td><td>1543</td><td></td><td>1534</td><td>vC=OH<sup>+</sup> (acetyl)</td></tr><tr><td>K</td><td>1612</td><td>1612</td><td>1617</td><td>1623</td><td></td><td>vC=N</td></tr><tr><td>L</td><td>1705</td><td></td><td></td><td></td><td>1695</td><td>vC=N</td></tr><tr><td>L'</td><td>1735</td><td></td><td></td><td>1727</td><td></td><td>vC=OH<sup>+</sup> (acetyl), vC=O (carboxyl)</td></tr><tr><td>M</td><td>1779</td><td>1781</td><td>1785</td><td></td><td>1789</td><td>vC=O (carboxyl)</td></tr></tbody></table>

<table><caption>**Table 2.** Experimental and calculated vibrational frequencies for the selected isomers NAcMetSOH⁺ at B3LYP/6-311+g(d,p) level of the theory, and corresponding band assignments.</caption><thead><tr><th></th><th>Experiment</th><th>acMOH<br>+ _2</th><th>acMOH<br>+ _3</th><th>Assignments</th></tr></thead><tbody><tr><td>A</td><td>728</td><td>744</td><td>734</td><td>vCC, vSC, δCNH, δCOH</td></tr><tr><td>B</td><td>862</td><td>859</td><td>842</td><td>vS=OH<sup>+</sup>, vCC</td></tr><tr><td>C</td><td>973</td><td>1000</td><td></td><td>vCC, δCOH, δCH<sub>2</sub></td></tr><tr><td>D</td><td>1143</td><td>1154</td><td>1163</td><td>vCO, δCOH, δCH<sub>2</sub></td></tr><tr><td>E</td><td>1235</td><td>1263</td><td>1262</td><td>vCC, vCN, δCNH, δCOH</td></tr><tr><td>F</td><td>1292</td><td>1296</td><td></td><td>vC=OH<sup>+</sup> (acetyl), δCOH, δCH<sub>2</sub></td></tr><tr><td>G</td><td>1349</td><td></td><td>1352</td><td>vCC, δCOH</td></tr><tr><td>H</td><td>1399</td><td></td><td>1393</td><td>vCC, δCH<sub>3</sub></td></tr><tr><td>I</td><td>1549</td><td>1518-<br>1578</td><td>1515-<br>1620</td><td>vCN, vC=OH<sup>+</sup> (acetyl),<br>vC=O (acetyl),</td></tr><tr><td>J</td><td>1698</td><td>1746</td><td></td><td>vCN</td></tr><tr><td>K</td><td>1785</td><td></td><td>1788</td><td>vC=O (carboxyl)</td></tr></tbody></table>

The experimental IRMPD spectrum of [NAcMet-H⁺]⁻ and the computed IR spectra for acMH-1, acMH-2, acMH-3 and acMH-4 structures optimized at the DFT level of theory are displayed in Figure 9 together with the corresponding 3D-geometries. All four calculated IR spectra show good agreement with the IRMPD spectrum.

acMH-2, acMH-3 and acMH-4 lie 2.7, 4.0 and 5.4 kJ/mol higher in energy compared to the most stable structure acMH-1, respectively. The four conformers have similar IR spectra and each of them can explain the ensemble of IR bands observed experimentally, so that they could be populated under our experimental conditions. All structures are stabilized by non-covalent NH...O interaction. The deprotonation site is on the carboxyl group. Other calculated structures at higher relative energy with their corresponding spectra are detailed in Figure SI10–11. The band assignment for [NAcMet-H⁺]⁻ is reported in Table 3.

Calculations have also been performed for the deprotonated oxo-form of NAcMet. The IRMPD spectrum of [NAcMet(O)-H⁺]⁻ is compared with the calculated IR spectra of

<table><caption>**Table 3.** Experimental and calculated vibrational frequencies for the selected isomers [NAcMet-H⁺]⁻ at B3LYP/6-311+g(d,p) level of the theory, and corresponding band assignments.</caption><thead><tr><th></th><th>Experiment</th><th>acMH-1</th><th>acMH-2</th><th>acMH-3</th><th>acMH-4</th><th>Assignments</th></tr></thead><tbody><tr><td>A</td><td>759</td><td>758</td><td>776</td><td>766</td><td>772</td><td>vSC, δCNH, δCH<sub>2</sub></td></tr><tr><td>B</td><td>839</td><td>834</td><td>832</td><td>857</td><td>854</td><td>vCC, vSC</td></tr><tr><td>C</td><td>952</td><td>959</td><td>960</td><td>964</td><td>964</td><td>vCC, vCN, δCH<sub>3</sub></td></tr><tr><td>D</td><td>1112</td><td>1112</td><td>1131</td><td>1109</td><td>1107</td><td>vCN, vCC, δCH<sub>2</sub></td></tr><tr><td>E</td><td>1165</td><td>1164</td><td>1208</td><td>1188</td><td>1186</td><td>vCN, vCC, δCH<sub>2</sub></td></tr><tr><td>F</td><td>1325</td><td>1327</td><td>1317</td><td>1309</td><td>1309</td><td>vCC, δ<sub>umb</sub>CH<sub>3</sub></td></tr><tr><td>G</td><td>1458</td><td>1480</td><td>1483</td><td>1483</td><td>1482</td><td>vCN, vCC, δCH<sub>3</sub></td></tr><tr><td>H</td><td>1662</td><td>1657</td><td>1653</td><td>1659</td><td>1660</td><td>vC=O (acetyl), vC=O (carboxyl)</td></tr></tbody></table>
<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>**Figure 8.** Comparison of the experimental IRMPD spectrum of NAcMet(O)H⁺ with convoluted calculated IR absorption spectra of representative conformers of the lowest-energy conformers optimized at the B3LYP/6-311++G(d,p) level of theory. The H-bond distances are reported in Å. The corresponding structures are reported on the right together with the relative Gibbs free energy (ΔG) at 298 K in kJ/mol, calculated at B3LYP/6-311++G(d,p) level of theory.</figcaption></figure>

the lowest energy conformers **acMOH-1**, **acMOH-2**, **acMOH-3** and **acMOH-4** in Figure 10. The deprotonation site is located on the carboxylic group. **acMOH-1** is the most stable structure for [NAcMetO-H⁺]⁻ and its calculated spectrum can justify all the bands observed in the experimental IRMPD spectrum. The structures **acMOH-2**, **acMOH-3** and **acMOH-4** are 2.9, 5.3 and 6.6 kJ/mol higher in energy compared to **acMOH-1**. They possess similar spectral features and are in good agreement

with the IRMPD spectrum as well. Hence, all of these low-energy structures could be populated under our experimental conditions. As for the deprotonated non-oxidized form, a C=O... H-N interaction stabilizes these low-energy geometries. Other structures explored with their corresponding spectra are detailed in Figure SI12-13.

The observed IR bands of [NAcMet(O)-H⁺]⁻ are tentatively assigned in Table 4. The diagnostic band of this oxo form, the
<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /><figcaption>**Figure 9.** Comparison of the experimental IRMPD spectrum of [NAcMet-H⁺]⁻ with convoluted calculated IR absorption spectra of representative conformers of the lowest energies geometries optimized at the B3LYP/6-311+g(d,p) level of theory. The H-bond distances are reported in Å. The corresponding structures are reported on the right together with the relative Gibbs free energy (ΔG) at 298 K in kJ/mol, calculated at B3LYP/6-311+g(d,p) level of theory, is provided in parentheses.</figcaption></figure>

intense band D centered at 1054 cm⁻¹, is assigned to the stretching mode of S=O bond coupled with CH₃ bending. It represents the major spectral difference between the non-oxidized and oxidized forms in negative mode. The most intense band G at 1665 cm⁻¹ is the C=O stretching in acetyl group and in carboxyl group.

As expected, the intense band observed at 1054 cm⁻¹ and corresponding to the S=O stretching mode is diagnostic to elucidate the oxidation site and the IRMPD results confirm that the oxidation of NAcMet leads to the formation of a sulfoxide group in the side chain, although the N-terminal group is blocked by an acetyl group. The study of these systems in negative mode allows to find the diagnostic band for the
<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /><figcaption>**Figure 10.** Comparison of the experimental IRMPD spectra of [NAcMet(O)-H⁺]⁻ with convoluted calculated IR absorption spectra of representative conformers of the lowest energy structures optimized at the B3LYP/6-311++g(d,p) level of theory. The H-bond distances are reported in Å. The corresponding structures are reported on the right together with the relative Gibbs free energy (ΔG) at 298 K in kJ/mol, calculated at B3LYP/6-311++g(d,p) level of theory.</figcaption></figure>

sulfoxide formation in the expected energy region around
1000 cm⁻¹.

Conclusions

In this work, terminal-blocked Met (NAcMet, NAcMetOCH₃) and
MetMet (NAcMetMetOCH₃) have been characterized by CID-MS²

analysis and IRMPD spectroscopy. Our objective was to identify
the final oxidation products of model peptide backbones
containing methionine and to elucidate their structures in the
gas phase.

In the MS spectra, similar oxidation products have been
observed for terminal blocked Met residues, products corre-
sponding to the oxygen addition (+16 Da and +32 Da) and to
dehydrogenation (-2 Da). However, little or no decarboxylation
<figure><img src="image_24.png" /></figure>

<table><caption>**Table 4.** Experimental and calculated vibrational frequencies for the selected isomers [NAcMetSO-H⁺]⁻ at B3LYP/6-311++g(d,p) level of the theory, and corresponding band assignments.</caption><thead><tr><th></th><th>Experiment</th><th>acMOH_1</th><th>acMOH_2</th><th>acMOH_3</th><th>acMOH_4</th><th>Assignments</th></tr></thead><tbody><tr><td>A</td><td>777</td><td>798</td><td></td><td>818</td><td>792</td><td>δCNH, δSCH</td></tr><tr><td>B</td><td>843</td><td>827</td><td>810</td><td>834</td><td>852</td><td>vCC, δSCH δCH<sub>2</sub></td></tr><tr><td>C</td><td>956</td><td>893</td><td></td><td></td><td></td><td>vCC, vSC, δCH<sub>2</sub></td></tr><tr><td>D</td><td>1054</td><td>1057</td><td>1059</td><td>1062</td><td>1053</td><td>vS=O, δCH<sub>3</sub></td></tr><tr><td>E</td><td>1323</td><td>1339</td><td>1319</td><td>1315</td><td>1329</td><td>vCC, δ<sub>umb</sub>CH<sub>3</sub></td></tr><tr><td>F</td><td>1470</td><td>1482</td><td>1485</td><td>1482</td><td>1478</td><td>vCN, δCNH, δCH<sub>3</sub></td></tr><tr><td>G</td><td>1665</td><td>1654</td><td>1648</td><td>1656</td><td>1658</td><td>vC=O (acetyl), vC=O (carboxyl)</td></tr></tbody></table>

has been observed after oxidation induced by gamma-radiolysis. These results agree with time-resolved studies performed on NAcMetOCH₃ *OH-induced oxidation. They indicate that the α-amino radicals (αC*), leading to the decarboxylation, have a short lifetime and a relatively low yield compared with other radical formations.[32] CID-MS² results confirmed the formation of a sulfoxide group after oxidation by revealing the diagnostic fragment corresponding to the loss of HSOCH₃ (–64 Da) from + 16 oxo-form. When several susceptible sulphur sites are present, the final oxidation products are a mixture of multiple oxo-forms.

Blocked model peptide backbones are interesting systems to investigate the effect of the neighbouring groups on the final oxidation products, but their IRMPD spectra in the fingerprint region are congested leading to a complicated assignment of the vibrational bands. IRMPD experiments supported by DFT calculations performed here for the simplest systems as models have shown that the diagnostic band for the sulfoxide group formation is red-shifted from ~1000 to 862 cm⁻¹ in the case of protonated oxidized NAcMet and NAcMetOCH₃, compared to protonated Met.[33] The diagnostic band for the sulfoxide formation in the IRMPD spectrum is, for NAcMet(O)H⁺, no longer the stretching mode of a neutral S=O group at ~1000 cm⁻¹ as in the case of MetSOH⁺, but the stretching mode of S=OH⁺ at 862 cm⁻¹. Protonation for NAcMetH⁺ and its oxo form occurs on the acetyl oxygen also or mainly on sulfoxide oxygen. Protonation was observed on the amino group for both MetH⁺ and MetSOH⁺. The change in protonation site shifts the spectral signature of the sulfoxide group in the IRMPD spectrum in a more congested region of the IR spectrum. Experiments performed in negative mode confirmed this behaviour.

## Funding Sources

Financial support from the French National network INFRANALYTICS (FR 3624 CNRS) for conducting the research is gratefully acknowledged. This work was supported by a French government grant managed by the Agence Nationale de la Recherche under the France 2030 investment plan, reference ANR-22-EXLU-0003 (PEPR LUMA). The research leading to these results has received funding from LASERLAB-EUROPE (grant agreement

no. 871124, European Union's Horizon 2020 research and innovation programme). We gratefully acknowledge the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) for the support experiments performed at the FELIX Laboratory.

## Acknowledgements

The mass spectrometry platform SMAS at the Institut de Chimie Physique (ICP) in Paris-Saclay University and B. Redlich in Radboud University are gratefully acknowledged. We also thank FELIX facility and the technical staff, in particular Victor Claessen, Marije Barel, Wouter Stumpel and Bryan Willemsen for their efficient assistance during the IRMPD experiments. DS thanks Erna Gouwens from FELIX office for her assistance.

## Conflict of Interests

The authors declare no conflict of interest.

## Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Manuscript received: September 21, 2024

Revised manuscript received: November 10, 2024

Accepted manuscript online: November 12, 2024

Version of record online: November 29, 2024