Dr Marwa Abdelmouleh

Dr Mathieu Lalande

Dr Violaine Vizcaino

Dr Thomas Schlathölter

Jean-Christophe Poully
email: poully@ganil.fr

Jean- Christophe Poully

J.-C Poully

Photoinduced Processes within Noncovalent Complexes Involved in Molecular Recognition

Keywords: Ionizing radiation, Biomolecular systems, Mass spectrometry, Gas phase, Fragmentation

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Introduction

Noncovalent interactions are essential in molecular physics and chemistry, notably regarding molecular assemblies. Describing and understanding these interactions therefore allows for instance controlling the properties of new materials, catalyzing chemical reactions or creating sensors allowing for the detection of gases, explosives or drugs. Molecular recognition of a receptor by a drug is needed for medical efficiency and specificity, the latter being crucial for avoiding side-effects. Drugreceptor binding is governed by noncovalent links such as Hbonds, charge-dipole, cation-π or π-π interactions, which can be revealed by structural techniques like NMR, X-ray crystallography or IR spectroscopy. These methods have also allowed visualizing the complementary structures of ligands and receptors, a striking example of the structure/function relationship in biology. Particular classes of systems, such as intrinsically disordered proteins, at first seemed to challenge this relationship when they were first discovered. Over the last decades, their study led to extension of the structure/function paradigm to the relationship between dynamical structure and biological function. For instance, specific binding of particular disordered proteins to their targets (other proteins, DNA, oligosaccharides…) can trigger a conformational change (the so-called "induced fit") that leads to a well-defined structure and activates the system. Proteins also allow repairing harmful damage due to oxidants, via for instance specific recognition of damaged regions of DNA by enzymes. Oxidants are ubiquitous in tissues of living organisms, and include endogenous agents such as ionizing radiation. Despite their relevance, little is known about radiation effects on noncovalent complexes involved in molecular recognition. Stisova et al. [1] have shown that gamma rays damage a subtype of the estrogen receptor protein and destabilize its complex with a DNA sequence. Bond cleavages within a protein-DNA complex have been observed by means of X-ray crystallography. [2] However, such condensed-phase investigations often suffer from a lack of knowledge about the stoichiometry of noncovalent complexes. Gas-phase mass spectrometry techniques, on the other hand, allow for complete control over the system under study. The combination of mass spectrometry with synchrotron radiation has for instance recently led to the identification of the binding site of a ligand to the intrinsically disordered protein IB5. [3] In the present study, we combine synchrotron radiation and mass spectrometry in order to investigate photoabsorption in isolated molecular noncovalent complexes. As a model system for specific molecular recognition, we choose the vancomycin-Ac2 L K D A D A noncovalent complex, denoted V+R in the following. Vancomycin is a last-resort antibiotic against infections caused by Gram-positive bacteria: when vancomycin specifically binds to a precursor of the main constituent of the bacterial cell wall, it prevents its renewal and leads to death of the bacterium. Previous studies have established that binding is enantiospecific: the C-terminal D A D A sequence is mandatory for high affinity between vancomycin and the receptor. [4] The native molecular interactions responsible for this behavior have been unraveled by structural investigation of the V+R complex in a crystal by X-ray diffraction. [5] The resulting structure is depicted in Fig. 1, emphasizing the H-bonds involving the CO2 -group of the receptor. By means of IR spectroscopy, this biologicallyactive structure has been found to be conserved for deprotonated complexes in the gas phase, [6] but not for protonated species, [7] in consistence with the results from early low-energy collision-induced dissociation experiments of Roepstorff and co-workers. [8] Indeed, in the protonated complex, the neutral carboxyl group of Ac2 L K D A D A strongly disturbs the intermolecular H-bonding network, causing it to leave the binding pocket of vancomycin and being linked to the disaccharide moiety. [7] In the following, we report experimental results from tandem mass spectrometry and synchrotron radiation, and describe the intrinsic processes triggered by photoabsorption in noncovalent molecular complexes involving vancomycin.

Results and Discussion

Protonated species

First, let us consider the mass spectrum as well as the evolution of the yields of cationic products of isolated doubly-protonated vancomycin [V+2H] 2+ after single VUV photon absorption in the energy range from 13 to 32 eV (cf. Fig. 2). At all photon energies, we observe a peak at m/z 483.7 that can be assigned to non-dissociative ionization (NDI) of the precursor ion, leading to [V+2H] 3+ . This is consistent with measured ionization energies of peptide dications, which have been found to be in the 10-12 eV range. [9] All the other peaks in the mass spectrum are due to fragment ions. At m/z 306 and 572.5, we find the complementary B0 + and Y0 2+ fragments formed after cleavage of the glycosidic bond separating the disaccharide moiety from the central phenyl ring (see Fig. 1). Cleavage of the bond linking the two saccharide groups (leading to B1 + and Y1 2+ product ions at m/z 144 and 653.5, respectively) is also observed. However, these peaks could also be due to peptide backbone fragmentation into c1 + and z1 2+ ions. The peak at m/z 100 can be assigned to the a1 + peptide backbone fragment ion. All these findings are consistent with the data reported previously [10] from collisioninduced dissociation (CID) experiments on gas-phase argon and helium at a relatively high kinetic energy of 4 keV that is sufficient for ionizing collisions. The yields of Y0 2+ and B0 + are almost identical from 13 to 16 eV, and show the same evolution all over the energy range (see Fig. 2), indicating that both are formed by photoionization-induced cleavage of the glycosidic bond. Besides, their yields have an extrapolated appearance energy of about 11 eV, which can be accounted for by a low activation energy for this cleavage. Interestingly, additional peaks corresponding to H2O and CO2 sequential losses from Y0 2+ are also observed. These peaks are probably due to further fragmentation of Y0 2+ after ionization and internal conversion, followed by intramolecular vibrational energy redistribution. Such neutral molecule losses after glycosidic bond cleavage have already been reported to occur in glycopeptides after CID and UV photoabsorption, and have been attributed to radicalmediated processes. [11,12] The latter have also been put forward to explain the loss of neutral molecules from side chains of peptides after photoionization, with low appearance energies of a few eV. [13] In excellent agreement, we observe a systematic 2 eV shift of the yields of H2O and CO2 sequential losses from Y0 2+ with respect to the Y0 2+ yield itself (see Fig. 2). To investigate the effects of complexation on photofragmentation, we studied the triply-protonated vancomycin dimer. Most of the peaks in the mass spectrum appearing in Fig. 3 can be assigned to the same species as for isolated vancomycin, with a noticeable exception: the peak at m/z 865, which corresponds to the triply-charged complex between vancomycin and the Y0 + fragment. This peak is due to loss of the B0 + fragment ion from vancomycin after ionization of the dimer, without cleavage of all noncovalent bonds linking the two molecules. This process has been reported for the triplyprotonated eremomycin (an antibiotic similar to vancomycin) dimer after electron-capture dissociation, where radical reactions dominate. [14] It probably implies an activation energy lower than the binding energy between the two vancomycin molecules. When looking at the crystal structure of the vancomycin dimer in Fig. 3, we can see that it is stabilized by four intermolecular Hbonds. Furthermore, the sugar group of each vancomycin is bound to the rest of the molecule only via the glycosidic bond. If this structure is conserved in the gas phase, photo-induced cleavage or the glycosidic bond can lead to the formation of the sugar radical cation (B0 + ) and the complementary [V+Y0] 3+ complex ion. Additional information can be obtained from the photon energy dependence of the relative partial ion yields of these species (cf. Fig. 3). First, as for isolated vancomycin, the almost identical yields of the [V+Y0] 3+ and B0 + ions support our assignment. Their linear rise from 14 to 19 eV shows that these fragments result from ionization of [V2+3H] 3+ , whose appearance energy has been extrapolated to about 12 eV, a value close to the ionization energy of triply-charged peptides, [9] but also of sugars (about 9 eV for neutral deoxyribose, [15] plus 3 eV to account for the 3+ charge state of the system, [9] which gives 12 eV). This indicates that ionization can take place either in the sugar or peptidic moieties, followed by glycosidic bond cleavage with low activation energy. The appearance energy of 12 eV is also very similar to the one obtained for isolated vancomycin (see Fig. 2), which indicates that dimerization hardly affects this process. In order to identify the species formed by ionization and fragmentation of the Ac2 L K D A D A receptor mimic (denoted R) in the mass spectra obtained by irradiation of V+R complexes, let us take a look at the results for the protonated R dimer after absorption of a single 18 eV photon of (cf. Fig. 4). We mostly observe singly-charged ions due to fragmentation of the peptide backbone (a1 + , a2 + , b1 + and b2 + ), but also an internal fragment at m/z 126 (Ksc + ), due to cleavage of two bonds close to the lysine residue. The b1 + and b2 + fragments have previously been observed by Z. Yang et al. [17] after surface-induced dissociation of [V+R+H] + , where ground-state processes after vibrational energy deposition dominate, as they do in low-energy CID. Furthermore, it has been demonstrated that the same mechanism is responsible for the presence of backbone fragments in the mass spectra of other protonated peptides ionized by one VUV photon. [18] Vibrational energy redistribution also induces intermolecular dissociation of the dimer, which accounts for the presence of the intact protonated monomer [R+H] + at m/z 373. Interestingly, a peak at m/z 417 is also present: we attribute it to a complex between [R+H] + and CO2. The latter would thus come from the other R peptide, possibly after ionization, H transfer from the CO2H group and dissociation. This scenario is supported by the presence of a peak at m/z 328, which corresponds to the ionized peptide lacking one CO2 molecule. More insight can be gained by investigating the structure of the protonated R dimer. We have performed a conformational search by replica-exchange molecular dynamics and the structure of the lowest-energy conformer is shown in Fig. 4. The CO2H group of neutral R is linked to [R+H] + by two H-bonds not involving the H atom. Moreover, the latter is H-bound to a C=O group of the same peptide, allowing for intramolecular transfer. These structural features are consistent with our mechanism. Therefore, the observation of CO2 attached to [R+H] + would mean that these non-covalent intermolecular bonds remain intact after covalent bond cleavage. This can be due to a very low appearance energy, possibly pointing to a radical-mediated process, akin to the case of electron detachment-induced CO2 loss from deprotonated peptides containing carboxylate moieties. [19] This is a second example of the influence of the molecular 3D structure on the processes induced by photoionization, after the vancomycin dimer. The mass spectrum of the doubly-protonated noncovalent complex between vancomycin and R after single photon absorption at 14 eV is shown in Fig. 5. The intact ionized species is not observed despite an ionization energy that is probably lower than 14 eV. The complex thus always fragments after photoabsorption, likely due to a low binding energy. We also detect doubly-charged vancomycin as well as singlycharged R, which are complementary fragments after ionization. Interestingly, R is observed in protonated form and not as a radical cation. Since in the complex, vancomycin and R have been reported to be doubly-protonated and neutral, respectively, [7] the presence of [R+H] + might be explained by intermolecular proton transfer from ionized vancomycin to neutral R and/or H transfer from vancomycin to ionized R. It has been previously shown that for amino acid clusters, proton transfer can be triggered by ionization. [20] Moreover, this process is supported by the evolution of the main channels as a function of photon energy (vide infra). Fragments originating from both R and vancomycin can be clearly identified and all of them also appear in the spectra of isolated vancomycin (cf. Fig. 2) or the R dimer (cf. Fig. 4). This can be explained by dissociation of the complex followed by fragmentation of vancomycin and/or R, due to internal energy transfer. Besides, H2O and CO2 sequential losses from Y0 2+ are much less intense than for isolated vancomycin (see Fig. 2), which supports their formation being due to vibrational energy transfer, this energy being now redistributed over more degrees of freedom. Interestingly, we observe a new peak at m/z 678 that is absent from all previous spectra. We attribute this peak to a complex between the B0 + fragment of vancomycin and neutral R (denoted [B0+R] + in the following). The complementary fragment would thus be Y0, which we detect in two charge states: 1+ and 2+. Observing [B0+R] + is consistent with the structure of the gas-phase complex that has been reported previously from infra-red multi-photon dissociation (IRMPD) spectroscopy. [7] Indeed, R is H-bound to the sugar moiety, implying that glycosidic bond cleavage occurs without dissociation of all noncovalent bonds to form [B0+R] + . To figure out if this process is triggered by ionization, as for the vancomycin dimer, let us look at the relative yields of [B0+R] + , Y0 2+ and Y0 + as a function of photon energy (cf. Fig. 5). The yields of [B0+R] + and Y0 2+ both increase smoothly between 14 and 18 eV, and their extrapolated appearance energy is 12 eV. The same behavior has been observed for the vancomycin dimer (see Fig. 3), suggesting the same underlying mechanism, namely ionization of vancomycin within the complex, followed by glycosidic bond cleavage with low activation energy. The yield of Y0 + does not correlate with that of [B0+R] + , which shows that they are probably formed by different channels. This further confirms the influence of geometrical structure on fragmentation of noncovalent complexes induced by photoionization. Even more information can be obtained by plotting the sum of yields of all fragments of R as well as vancomycin, along with those of intact R and vancomycin (cf. Fig. 5). The most striking is the opposite trends for the sum of vancomycin and R fragments between 14 and 20 eV and their crossing around 15 eV. Fragmentation of R and vancomycin dominate at low and high energy, respectively. The yield of intact protonated receptor follows a trend opposite to that of the receptor fragments, but the sum of their yields is not constant, which means that all fragments do not come from the protonated R. We attribute the complementary amount to ionization of neutral R within the complex, followed by fragmentation. This process would thus be quenched by ionization-induced proton transfer between vancomycin and R, followed by dissociation of the complex and release of [R+H] + , whose yield is rising between 14 and 20 eV, as seen above for ionization-induced species from vancomycin monomer and dimer. Further increasing photon energy leads to a slight decrease of the yield of intact [R+H] + , attributed to its fragmentation, and consistently compensated by an increase of the sum of all yields of R fragments.

Deprotonated species

In order to probe the effects of charge state and structure on the photoabsorption of V+R noncovalent complexes, doublydeprotonated species have been studied, the results being shown in Fig. 6. The most intense peaks in the mass spectrum of doubly-deprotonated vancomycin after absorption of one 16 eV photon are attributed to loss of CO2 after photodetachment, forming the [V-CO2] -radical anion, and to the C0 -fragment due to cleavage of the bond linking the central phenyl ring to the oxygen atom bound to the disaccharide group. Minor ions Y1 - and Y0 --CO2 are formed by glycosidic bond cleavage. The mass spectra of [V+R-2H] 2-also contain the same intense peak corresponding to [V-CO2] -, and the other intense peak is assigned to [V+R-2H-CO2] -. Interestingly, the latter fragment means that CO2 loss is observed in the complex without breakage of all noncovalent bonds between vancomycin and R (see Fig. 1). It is thus clear that the most abundant channel triggered by single VUV photoabsorption in deprotonated species is loss of CO2, which is fundamentally different from the case of protonated systems. CO2 loss induced by excitation or detachment of one electron from valence orbitals has been previously reported for deprotonated peptides. [21][22][23] This has been rationalized by electron transfer or detachment from CO2 -, followed by a radical reaction and CO2 loss, [24] and is perfectly plausible in deprotonated vancomycin as well as [V+R-2H] 2- since they contain one and two CO2 -groups, respectively, according to previous IR spectroscopy studies. [6] Therefore, electron detachment from CO2 -can explain the observed CO2 loss. In [V+R-2H] 2-, there is one CO2 -in R and one in vancomycin (see Fig. 1), electron detachment can thus occur from R or vancomycin. The other major peak in the mass spectrum of the complex (see Fig. 6) can be assigned to vancomycin without CO2, which can be interpreted as a consequence of electron detachment from R followed by vibrational energy transfer and dissociation of the complex, together with CO2 loss from deprotonated vancomycin, due to excess vibrational energy. This is supported by experimental results from low-energy collision-induced dissociation [8] and IRMPD of deprotonated V+R complexes, [6] where processes are triggered by vibrational energy deposition. In the mass spectra of the complex after photoabsorption, two peaks corresponding to deprotonated R and CO2 loss are also observed (see Fig. 6): BOTH are most probably due to dissociation of the complex after electron detachment from vancomycin followed by vibrational energy transfer. Their weak intensity points to a higher binding energy of the complex after electron detachment from vancomycin than from R. This is consistent with the structure of the precursor complex in the gas phase, which has been shown to be the native structure with strong H-bonds linking the CO2 -group of R to the binding pocket of vancomycin (see Fig. 1). [6] After electron detachment from R, these chargedipole interactions do not exist anymore, thus decreasing the complex binding energy, whereas little structural effect is expected after electron detachment from the CO2 -group of vancomycin, because it is not bound to R, and thus can leave the complex without breaking it, leading to [V+R-2H-CO2] -. The influence of the native structure is also supported by the much lower intensity of the peak attributed to further loss of CO2, leading to [V+R-2H-2CO2] -: when both CO2 groups are lost, it mostly leads to dissociation of the complex. When photon energy rises from 16 to 24 eV (cf. Fig. 6), we observe an increase of [V-H-CO2] -and [R-H-CO2] -. In this energy range, we have previously demonstrated that the amount of transferred vibrational energy increases, [13,25] it is therefore in line with our hypothesis of [V-H-CO2] -and [R-H-CO2] -being due to internal conversion and intramolecular vibrational energy redistribution after electron detachment. It is insightful to compare our results on complexes of vancomycin and Ac2 L K D A D A receptor mimic to those obtained by Roepstorff and co-workers: [8] they have shown that CID at 70 eV kinetic energy in the laboratory frame leads to the same fragmentation yield for doubly-deprotonated as for doublyprotonated complexes. The fact that our results indicate that VUV photoabsorption induces less fragmentation for the doublydeprotonated complex might come from the radical species created after electron removal: radical-mediated processes have been shown to play an important role, both for protonated and deprotonated peptides for instance. [13,19,26] In low-energy CID, these processes are negligible for closed-shell species and instead, charge-remote or charge-directed mechanisms are dominant. [27] Therefore, one can expect different kinetics and energetics between radical and closed-shell species.

Conclusions

In this article, we have shown that photoabsorption of vancomycin within isolated noncovalent complexes leads to very different products depending on the initial charge state: for protonated species, the glycosidic bond is cleaved with low activation energy, in contrast to the deprotonated species containing carboxylate moieties that undergo CO2 loss and intermolecular dissociation. A competition between dissociative ionization and ionization-induced intermolecular proton transfer is observed for the doubly-protonated vancomycin-Ac2 L K D A D A complex. An influence of geometrical structure is also observed in all noncovalent complexes investigated. Furthermore, we show that loss of vancomycin-Ac2 L K D A D A noncovalent interactions after single electron removal occurs to a much lower extent when the native structure of the complex is retained. Further work is needed to know if the receptor moves away from the binding pocket of vancomycin after photoabsorption: this structural change could be investigated by tandem ion mobility spectrometry. Such studies have already been successfully performed on deprotonated eosin Y.

[28]

Experimental and Theoretical Section

Vancomycin hydrochloride and Ac2 L K D A D A were purchased from Sigma-Aldrich as powders of over 80 % purity, and were used without further purification. Solution were prepared in 50:50 (volume ratio) water/methanol at 50 μM concentration with 1 % of formic acid for protonated species and potassium hydroxide for deprotonated species.

A home built tandem mass spectrometer, described in detail elsewhere, [29] has been used to record mass spectra of the ionic photoproducts from the interaction between molecular systems and synchrotron radiation. Briefly, protonated or deprotonated molecular systems are produced with an electrospray ion source and transported into the vacuum chamber through a heated capillary. The molecular ion beam is then focused into an ion funnel and guided into an octopole before being mass-over-charge selected with a quadrupole mass-filter and subsequently accumulated in a 3D radiofrequency ion trap. Trapping is facilitated by collisions with a helium buffer gas injected into the ion trap during the filling process. Molecular ions have been irradiated by VUV photons at the U125-2_NIM [START_REF]für Materialien und Energie[END_REF] beamline of the BESSY II synchrotron (Helmholtz-Zentrum Berlin). Photon beam exposure of the trap content, typically during 300 ms to 1000 ms, is controlled with a mechanical shutter in order to guarantee that more than 90 % of the product cations result from the absorption of a single photon. To do so, the irradiation time is tuned to induce a depletion of the precursor peak below 10 %. Since the absorption of multiple photons is a sequential process at these fluxes (10 12-13 s -1 ), the absorption events are independent, thus a probability 𝑝 for absorbing one photon gives the probability 𝑝 2 for two photons. Neglecting the absorption of more than two photons, we obtain 𝑝 2 + 𝑝 < 0.1 and thus 𝑝 < 0.09. Precursor ions and cationic fragments are then extracted from the trap and analysed by time-of-flight mass spectrometry. Mass spectra of the non-irradiated trap content and irradiated residual gas are recorded as well in order to spot background peaks due to photoionization of residual gas molecules.

Then, the beam-on mass spectrum is subtracted from the beam-off one, and the resulting spectrum shows the precursor depletion with a negative intensity. Assuming that absorption of one photon leads to ionisation and/or fragmentation of the precursor ion, this depletion is proportional to the total photo-absorption yield. All mass spectra are normalised by the precursor depletion, as well as by the sum of the abundances of all ions formed by photoabsorption.

Replica-exchange molecular dynamics have been performed for the protonated Ac2 L K D A D A dimer to get insight into its geometrical structure. This method has already been used in order to find the structure of the lowest-energy conformations of [V+R+2H] 2+ , because it ensures an efficient sampling of the potential energy surface of a molecular system,. [7] The chosen protonation site of [R+H] + within the dimer is the oxygen atom of the lysine side-chain acetyl group, which substitutes the NH2 group that would have had the highest proton affinity in the molecule. We used the AMBER99 force field and 30 replicas were sampled from 20 to 600 K with a geometric progression. Each replica was propagated for 20 ps for thermalisation to occur, and then for an additional 2 ns with a 1 fs time step. For thermal equilibrium, we used a Berendsen thermostat with a 0.1 ps -1 coupling constant. Exchange between neighboring replicas was attempted every 100 fs. For each replica, a snapshot was taken every 40 ps and the geometry of the conformers obtained was further optimized with the AMBER99 force field. The structure of the lowestenergy conformer has further been optimized at the B3LYP/6-31g(d) level thanks to the Gaussian09 software. [START_REF] Frisch | Gaussian 09[END_REF]

Figure 1 .

 1 Figure 1. Chemical structure of the V+R complex in the condensed phase, showing native intermolecular H-bonds as dashed lines. The most basic and acidic sites are protonated and deprotonated, and encircled in blue and orange, respectively. The usual nomenclature for peptide backbone as well as oligosaccharide fragmentation is used for the main fragments observed after photoabsorption. Green arrows indicate on which side of the cleaved bond the charge is located.

Figure 2 .

 2 Figure 2. Top: mass spectrum of [V+2H] 2+ after absorption of one 20 eV photon (the usual nomenclature for fragmentation of peptides and oligosaccharides has been used and NDI stands for non-dissociative ionization); bottom: relative yield of photo-induced channels of [V+2H] 2+ as a function of photon energy, normalized by detector efficiency and the sum of all peak areas (see the experimental section in SI). The green dashed line is here to guide the eye.

Figure 3 .

 3 Figure 3. Top: mass spectrum of [V2+3H] 3+ (m/z 967) after absorption of one 14 eV photon. The same nomenclature as in Fig. 2 has been used for fragments. The bond cleavages accounting for their formation are indicated in Fig. 1. Middle: evolution of the yields of [V+Y0] 3+ and B0 + after single photoabsorption of [V2+3H] 3+ as a function of photon energy, normalized by the detector efficiency and the sum of all peak areas (see the experimental section in SI). The dashed lines are here to guide the eye. Bottom: Geometrical structure of the vancomycin dimer in a crystal

[ 16 ]

 16 (PDB 1AA5), with intermolecular H-bonds depicted in dashes and each molecule in a different color. The sugar groups are encircled in green.

Figure 4 .

 4 Figure 4. Top: mass spectrum of [R2+H] + (m/z 745) after absorption of one 18 eV photon. The same nomenclature as in Fig. 2 has been used for fragments. The bond cleavages accounting for their formation are indicated in Fig. 1. Bottom: structure of the lowest-energy conformer after REMD, optimized at the B3LYP/6-31g(d) level (see the "Experimental set-up and simulations" section for details). Intermolecular H-bonds are depicted with red dashes. The intramolecular H-bond involving the transferred H (see text) is highlighted in green dashes. C, N, O and H atoms are colored in grey, blue, red and white, respectively. The proton is encircled in blue.

Figure 5 .

 5 Figure 5. Top: mass spectrum of [V+R+2H] 2+ after absorption of one 14 eV photon. The same nomenclature as in Fig. 2 has been used for fragments. The bond cleavages accounting for their formation are indicated in Fig. 1. Middle and bottom: relative yield of the main species formed after single photoabsorption of [V+R+2H] 2+ as a function of photon energy, normalized by the detector efficiency and the sum of all peak areas (see the experimental section in SI). The dashed lines are here to guide the eye. The yields of the intact protonated receptor and intact vancomycin are indicated by "Receptor" and "Vancomycin", respectively.

Figure 6 .

 6 Figure 6. Mass spectra of [V-2H] 2-(top) and [V+R-2H] 2-(bottom) after absorption of one VUV photon. Loss of one and two CO2 molecules from the latter after electron detachment is denoted -CO2 (m/z 1775) and -2CO2 (m/z 1731), respectively. The same nomenclature as in Fig. 2 has been used for fragments. The bond cleavages accounting for their formation are indicated in Fig. 1.

Acknowledgements

We thank Helmholtz Zentrum Berlin for the allocation of synchrotron radiation beamtime, P. Baumgärtel for his support during experiments, and the French "Conseil Régional de Normandie" for a PhD funding (#15P01339). This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 730872.


Marwa Abdelmouleh

Mathieu Lalande

Johnny El Feghaly

Violaine Vizcaino

André Rebelo

Thomas Schlathölter

Jean-Christophe Poully
email: poully@ganil.fr

Samuel Eden

Mass spectral signatures of complex post-translational modifications in proteins: a proof-of-principle based on X-ray irradiated vancomycin

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INTRODUCTION

Post-translational modifications (PTM) of proteins are essential in biology, since they tailor protein activity by cleavage and/or creation of covalent bonds. The removed and/or added chemical groups can be quite diverse: the most common PTM are phosphorylation, acetylation and glycosylation. They involve the attachment of a phosphoryl, an acetyl or a carbohydrate group, respectively, to an amino-acid side chain. Other important PTM lead to removal of a peptide from the protein N-or C-terminal side, for instance in collagen. Hydroxylation of prolines and lysines is another PTM, which for instance stabilizes the collagen triple-helix structure responsible for the specific mechanical properties of connective tissues such as skin, cartilage, nails and bones. PTM can also result from oxidative stress, aromatic and sulfur-containing amino-acids being especially prone to cross-linking or oxidation. [START_REF] Hawkins | Detection, Identification, and Quantification of Oxidative Protein Modifications[END_REF] For instance, these processes are crucial for the synthesis of glycopeptide antibiotics such as vancomycin. The latter is a lastresort drug against Gram-positive bacteria, blocking the renewal of their cell-wall by acting as a ligand for a precursor of the main cell-wall component. Vancomycin is naturally formed by bacteria but has also been synthesized. [START_REF] Nicolaou | Total Synthesis of Vancomycin[END_REF] Once the seven-residue peptidic chain has been created, several PTM have to occur in the five tyrosine side-chains. They involve the addition of OH groups, cross-linking, the addition of chlorine atoms to two of these side-chains, and glycosylation of the central one (see Fig. 1). The numerous and dense cross-linkages in the resultant peptide create a rigid binding pocket allowing for stereospecific recognition of the receptor via noncovalent binding. Identifying such a complex modification is extremely challenging, even for state-of-the-art methods such as capillary electrophoresis or mass spectrometry (MS) techniques. [START_REF] Doll | Mass Spectrometry-Based Detection and Assignment of Protein Posttranslational Modifications[END_REF] The latter have proven to be very powerful for deciphering the site and identity of PTM in proteins such as histones, which undergo a particularly large number of PTM. [START_REF] Huang | Quantitative Proteomic Analysis of Histone Modifications[END_REF] Generally, two approaches can be followed: top-down or bottom-up. In the first, gas-phase intact proteins are cleaved into fragments whose mass-over-charge (m/z) ratio allows identifying PTM. In the second, proteins undergo enzymatic digestion that produces peptides, which are identified thanks to MS and fragmentation techniques. Protein and peptide fragmentation is often performed by collision-induced dissociation (CID) using a rare gas or N 2 , and when coupled to tandem MS, CID has allowed identifying disulfide bridges in conotoxins. [START_REF] Gupta | Disulfide Bond Assignments by Mass Spectrometry of Native Natural Peptides: Cysteine Pairing in Disulfide Bonded Conotoxins[END_REF] However, electron-capture or transfer dissociation are better suited for assigning labile PTM such as Olinked glycosylation or phosphorylation of serines or threonines. [START_REF] Doll | Mass Spectrometry-Based Detection and Assignment of Protein Posttranslational Modifications[END_REF] A combination of electron-transfer dissociation and CID has been applied for studying cyclotides containing numerous cysteine knots. [START_REF] Foreman | Gas-Phase Sequencing of Cyclotides: Introduction of Selective Ring Opening at Dehydroalanine via Ion/Ion Reaction[END_REF] VUV photoionization has recently been proposed for the identification of multiple PTM in histones. [START_REF] Greer | Top-Down Characterization of Heavily Modified Histones Using 193 Nm Ultraviolet Photodissociation Mass Spectrometry[END_REF][START_REF] Greer | Extensive Characterization of Heavily Modified Histone Tails by 193 Nm Ultraviolet Photodissociation Mass Spectrometry via a Middle-Down Strategy[END_REF] In 2019, photodissociation using a UV laser revealed mass spectral signatures of dityrosine cross-linking in peptide dimers. 9 In this article, we report the proof-of-principle that soft Xray photoionization, as a biomolecular fragmentation technique, gives robust mass spectral signatures of particularly complex and dense PTM. Using vancomycin as a model heavily-modified peptide, we prove that all PTM, not only labile ones, can be identified with mass spectrometry. Our work extends the potential of mass spectrometry techniques in the growing field of PTM identification and quantitation in proteins.

EXPERIMENTAL

Vancomycin hydrochloride and Ac 2 L K D A D A have been purchased from Sigma-Aldrich as powders of over 80 % purity, and used without further purification. Solutions have been prepared in 50:50 (volume ratio) water/methanol at 50 μM concentration with 1 % of formic acid to protonate the molecules.

A home built tandem mass spectrometer, described in detail elsewhere, [START_REF] Bari | Photodissociation of Protonated Leucine-Enkephalin in the VUV Range of 8-40 EV[END_REF] has been used to record mass spectra of the ionic photo-products from the interaction between molecular systems and synchrotron radiation. Briefly, protonated molecular systems are produced with an electrospray ion source and transported into the vacuum chamber through a heated capillary. The molecular ion beam is then focused into an ion funnel and guided into an octopole before being mass-over-charge selected with a quadrupole mass-filter (QMF) and subsequently accumulated in a 3D radiofrequency ion Paul trap. Trapping is facilitated by collisions with a helium buffer gas injected into the ion trap during the filling process. Molecular ions have been irradiated by X-ray photons at the U49-2 PGM-1 beamline of the BESSY II synchrotron (Helmholtz-Zentrum Berlin). Photon beam exposure of the trap content, typically during 300 to 1000 ms, is controlled with a mechanical shutter in order to guarantee that more than 90 % of the product cations result from the absorption of a single photon. To do so, the irradiation time is tuned to induce a depletion of the precursor ion below 10 %. Since the absorption of multiple photons is a sequential process at these fluxes (10 12-13 s -1 ), the absorption events are independent, thus a probability 𝑝 for absorbing one photon gives the probability 𝑝 2 for two photons. Neglecting the absorption of more than two photons, we obtain 𝑝 2 + 𝑝 < 0.1 and thus 𝑝 < 0.09. Precursor ions and cationic fragments are then extracted from the trap, analyzed by a time-of-flight reflectron mass spectrometer, and detected by microchannel plates. Mass spectra of the non-irradiated trap content (beamoff) and irradiated residual gas are recorded as well, the latter allowing to spot background peaks due to photoionization of residual gas molecules. Then, the beam-on mass spectrum is subtracted from the beam-off one, and the resulting spectrum shows the precursor ion depletion with a negative intensity.

Assuming that absorption of one photon leads to ionization and/or fragmentation of the precursor ion, this depletion (area under the peak) is proportional to the total photo-absorption yield. All relative yields have been obtained by calculating the area under each peak, normalizing by the precursor ion depletion, by the total yield of all cationic species formed by photoabsorption, and by detection efficiency. 11

RESULTS AND DISCUSSION

The soft X-ray photoabsorption spectra of doublyprotonated vancomycin [V+2H] 2+ are shown in Fig. 2. In the spectrum at 100 eV, we mainly observe the same species as we have previously observed after photoabsorption in the VUV range. [START_REF] Abdelmouleh | Photoinduced Processes within Noncovalent Complexes Involved in Molecular Recognition[END_REF] The peak at m/z 483.8 corresponds to nondissociative ionization (NDI) of the precursor ion, a minor process compared to fragmentation following ionization. The yields of the complementary 𝑌 0 2+ and 𝐵 0 + fragments are significantly higher (see Fig. 2). Both are due to cleavage of the glycosidic bond (linking the carbohydrate group to the rest of the molecule) after ionization. X-ray photoionization thus allows identifying the mass of the carbohydrate involved in glycosylation. The most intense peaks are assigned to the 𝑎 1 + and 𝑐 1 + fragments of the pseudo-peptidic backbone (see Fig. 1). Note that the peak corresponding to the latter (at m/z 144) can also be attributed to 𝐵 1 + , formed by cleavage of the glycosidic bond within the carbohydrate moiety. The relative yield of NDI and large fragments is smaller than in the case of photons in the 14-30 eV range, [START_REF] Abdelmouleh | Photoinduced Processes within Noncovalent Complexes Involved in Molecular Recognition[END_REF] which can be explained by a rise of the vibrational energy transferred by the photon to the molecular system with photon energy, as it has previously been shown in the soft X-ray range. [START_REF] Schwob | Single-Photon Absorption of Isolated Collagen Mimetic Peptides and Triple-Helix Models in the VUV-X Energy Range[END_REF] With increasing photon energy, a more striking difference develops in the mass spectra. Whereas VUV photoabsorption solely leads to fragmentation into peaks at m/z 100, 118, 127, 144, 149, a multitude of additional peaks with m/z < 300 are induced by soft X-ray photoabsorption (see Fig. 2). In the following, we will show that these peaks can be assigned to internal fragments whose formation requires at least two bond cleavages (see below). The total relative yield of these internal fragments increases with photon energy up to 300 eV, and is compensated by the fall of non-dissociative ionization and all other fragments, with the exception of 𝑎 1 + (see Fig. 2). From 100 to 300 eV, the relative cross section for inner-valence orbital photoabsorption in-creases, and thus so does the average electronic excitation. After internal conversion to the electronic ground state and vibrational energy redistribution, this leads to more fragmentation as photon energy rises. This is consistent with the mechanism we proposed in our previous studies on protonated biologically-relevant molecules. [START_REF] Schwob | Single-Photon Absorption of Isolated Collagen Mimetic Peptides and Triple-Helix Models in the VUV-X Energy Range[END_REF][START_REF] Egorov | Near Edge X-Ray Absorption Mass Spectrometry of Gas Phase Proteins: The Influence of Protein Size[END_REF][START_REF] Egorov | A Comparative VUV Absorption Mass-Spectroscopy Study on Protonated Peptides of Different Size[END_REF] Figure 2. Top: mass spectra of [V+2H] 2+ after single photoabsorption between 100 and 531.5 eV. The position of the precursor ion (m/z 725.6) is represented by a purple line, the peak being negative (see the experimental section for details). The usual nomenclature is used for the peptide backbone as well as for oligosaccharide fragmentation and hence for the main fragments observed after photoabsorption (cf. Fig. 1). Internal fragments formed by at least two bond cleavages are indicated. Bottom: relative yield of the species formed after single photoabsorption as a function of photon energy, normalized by the detector efficiency and the total yield of photoinduced cations. NDI stands for non-dissociative ionization.

A deeper analysis can be achieved by zooming in the m/z 158 -278 region of the [V+2H] 2+ spectra, as can be seen in Fig. 3. Interestingly, none of these fragment ions have been observed after collision-induced dissociation (CID) at low energy (on the order of 10 eV), [START_REF] Jørgensen | Collision-Induced Dissociation of Noncovalent Complexes between Vancomycin Antibiotics and Peptide Ligand Stereoisomers: Evidence for Molecular Recognition in the Gas Phase[END_REF] where ionization does not occur. Similar groups of peaks do appear in the mass spectrum of singly-protonated vancomycin reported after CID at 4 keV kinetic energy on argon, but are not mentioned and their exact mass is not possible to obtain from the figure. [START_REF] Florencio | GAS EFFECTS IN THE COLLISION-INDUCED DECOMPOSITION OF PROTONATED AND CATIONIZED MOLECULES OF CARBOHYDRATE ANTIBIOTICS[END_REF] Since these high-energy collisions induce ionization, like X-rays, this seems to be consistent with these peaks coming from fragments being formed after ionization. In Fig. 3, one can notice that for soft X-ray photoabsorption, groups of peaks of similar intensity appear: they are separated by one mass unit, indicating singly-charged fragments. All precursor ions containing isotopes were present in the trap after m/z selection by the QMF (see the experimental section), but even considering the maximum number of carbon and chlorine atoms possible (22 carbons or 16 carbons and 2 chlorines) for the fragment corresponding to the peak of highest mass (270 amu), the natural abundance of [START_REF] Egorov | Near Edge X-Ray Absorption Mass Spectrometry of Gas Phase Proteins: The Influence of Protein Size[END_REF] C and 37 Cl cannot explain these patterns. Thus, they are probably due to extensive H scrambling, which has also been found to occur in photoionized 3-aminophenol, [START_REF] Bockova | Mapping the Complex Metastable Fragmentation Pathways of Excited 3-Aminophenol(+)[END_REF] an aromatic molecule containing one phenyl ring with one OH group. Indeed, these groups of peaks are typical of mass spectra of neutral H-rich molecules containing aromatic rings, e.g. ionized by electron impact, 18 ion impact at keV or MeV energy, [19][START_REF] Alvarado | Quantification of Ion-Induced Molecular Fragmentation of Isolated 2-Deoxy-D-Ribose Molecules[END_REF][START_REF] Poully | Formation and Fragmentation of Protonated Molecules after Ionization of Amino Acid and Lactic Acid Clusters by Collision with Ions in the Gas Phase[END_REF][START_REF] Agnihotri | Ionization of Uracil in Collisions with Highly Charged Carbon and Oxygen Ions of Energy 100 KeV to 78 MeV[END_REF] or by absorption of X-ray photons. [START_REF] Itälä | A Comparative Study of Dissociation of Thymidine Molecules Following Valence or Core Photoionization[END_REF][START_REF] Itälä | Fragmentation Patterns of Core-Ionized Thymine and 5-Bromouracil[END_REF] A good example is ionization of lorazepam, a molecule containing two aromatic rings substituted by one chlorine atom as in vancomycin: groups of peaks separated by 1 mass unit are also observed in the same mass range. 18 Ionization of small carbohydrates (such as that of vancomycin) also yields such groups of peaks, but in a much lower mass range, typically below 100 amu. [START_REF] Ptasinska | Inelastic Electron Interaction (Attachment/Ionization) with Deoxyribose[END_REF][START_REF] Vall-Ilosera | Photofragmen-tation of 2-Deoxy-D-Ribose Molecules in the Gas Phase[END_REF][START_REF] Shin | Extreme Ultraviolet Photoionization of Aldoses and Ketoses[END_REF][START_REF] Ghosh | A VUV Photoionization and Ab Initio Determination of the Ionization Energy of a Gas-Phase Sugar (Deoxyribose)[END_REF][START_REF] Shin | Vacuum Ultraviolet Photoionization of Carbohydrates and Nucleotides[END_REF] Therefore, we have calculated the mass of potential fragments originating from the groups containing aromatic rings in vancomycin. The results are shown in Scheme 1, where structures of fragments accounting for all groups of peaks seen in the mass spectra are proposed. In Fig. 3, we include vertical bars at the m/z of these singly-charged fragments with the maximum number of H atoms (leaving the bond orders unchanged), and without any H atom left. These bars mark off each group of peaks. To confirm that the natural abundance of [START_REF] Egorov | Near Edge X-Ray Absorption Mass Spectrometry of Gas Phase Proteins: The Influence of Protein Size[END_REF] C and 37 Cl isotopes does not account for the observed peaks, we also show the simulated isotopic pattern of each fragment in Fig. S1 (see the Supporting Information). It demonstrates that extensive H atom scrambling occurs for these fragments. Interestingly, their formation can be explained by only three pathways, two of them involving the central group containing the three phenyl rings (noted 1 in Fig. 1) and accounting for most of the groups of peaks. The remaining groups are assigned to fragments formed by a third pathway, starting with separation of the biphenol group bound to the C-terminus of the pseudo-peptidic backbone (noted 2 in Fig. 1). In addition, each aromatic ring can be identified separately. Thus, these groups of peaks assigned to internal fragments can be considered as a mass spectral signature of the complex PTM pattern of vancomycin. Internal fragments are not often exploited in MS of biopolymers, because of the intricate data analysis required. Here, the latter is made easier by the following features: the peaks fall at integer m/z values, are easily separated even with a modest resolving power, and are structured in distinct groups of peaks separated by 1 amu. Another important point is the influence of photon energy on this mass spectral signature: it can be deduced from Fig. 3. Despite the large energy range (100-531.5 eV) covering C, N and O K-edges, the relative intensity of these peaks varies only very slightly, the overall shape of the groups of peaks remaining remarkably stable. This is very important if they are used as mass spectral signature for identifying PTM. Consider also that we have shown in the previous paragraph that the total yield of these fragments increases with photon energy from 100 to 300 eV because of the increase in vibrational energy transferred to vancomycin after ionization. Therefore, this behavior might be traced to a high potential energy barrier for creating the largest fragments shown in Scheme 1, due to the need to cleave several bonds, and lower barriers separating the subsequent smaller fragments. The next question is to know whether the mass spectral signature of the PTM pattern of vancomycin is sensitive to a noncovalently bound molecular environment. To tackle this question, we have studied the vancomycin dimer as well as complexes between vancomycin and Ac 2 L K D A D A (K is lysine and A alanine), a peptidic model of its receptor, abbreviated R in the following. The chemical structure of the receptor can be found in Fig. S2. In all cases, absorption of one X-ray photon in the 100-531.5 eV range leads to ionization and fragmentation of the precursor molecular system. For instance, Fig. 4 shows that at 100 eV, the [V+R+2H] 2+ complex dissociates and subsequent intramolecular fragmentation of vancomycin and R occurs. Fragments of the latter have been identified by comparison with the mass spectra of [R 2 +H] + (see Fig. S2). The same fragments as for VUV photons are observed, [START_REF] Abdelmouleh | Photoinduced Processes within Noncovalent Complexes Involved in Molecular Recognition[END_REF] but large fragments are less abundant, as in the case of isolated vancomycin (cf. Fig. 2). Small fragments rise with photon energy from 100 to 300 eV, the total fragmentation yield slightly increasing for both vancomycin and R. Interestingly, the fragmentation yield of vancomycin is compensated by the falling yield of [R+H] + and vice versa, which is unexpected. In our previous study, we have attributed the formation of [R+H] + to a proton transfer between ionized vancomycin and neutral R. [START_REF] Abdelmouleh | Photoinduced Processes within Noncovalent Complexes Involved in Molecular Recognition[END_REF] Therefore, the decrease in the [R+H] + yield while vancomycin fragmentation increases might indicate that proton transfer becomes progressively less likely as photon energy increases. This might be due to more vibrational excitation as the X-ray photon energy rises from 100 to 300 eV, leading to the noncovalent complex dissociating on a faster timescale and quenching proton transfer. This is plausible because MacAleese et al. [START_REF] Macaleese | Sequential Proton Coupled Electron Transfer (PCET): Dynamics Observed over 8 Orders of Magnitude in Time[END_REF] have reported that proton transfer can take up to hundreds of microseconds in an ionized peptide radical cation. These results show that photon energies over 300 eV give the highest yield of vancomycin fragments. Among the latter, and for all noncovalent systems studied here ([V+R+2H] 2+ , [V+2R+2H] 2+ and [V 2 +3H] 3+ ), we observe the same internal fragments as for isolated vancomycin, their relative yield increasing with photon energy up to 300 eV (see Fig. 4 and S2-S4). We can also notice that whatever the noncovalent system, the overall relative yield of the internal fragments of vancomycin is lower than in the case of isolated vancomycin. This is consistent with our hypothesis of fragmentation in the ground-state due to vibrational energy transfer, this energy being redistributed into more degrees of freedom when the system gets larger. Furthermore, if we focus on the pattern made by the peaks attributed to these internal fragments, we observe a high similarity whatever the molecular environment (cf. Fig. 5). This indicates that the mass spectral signature is robust and can be used to identify the groups responsible for the fragments, and thus the corresponding post-translational modifications in proteins.

CONCLUSIONS

In this contribution, we have shown that X-ray photoabsorption of a highly modified tyrosine-rich peptide coupled to mass spectrometric analysis of the resulting fragments gives a signature that can be employed to characterize particularly complex and dense PTM, especially those resulting from oxidative stress. Indeed, this mass spectral signature is robust with respect not only to the X-ray photon energy over a large range (100-531.5 eV), but also to the molecular environment of the peptide. This proof-of-principle expands the potential of mass spectrometry techniques in proteomics. Further work is now required to test the applicability of this method to other peptides, proteins and a wider range of PTM.

Figure 1 .

 1 Figure 1. Chemical structure of vancomycin (C 66 H 75 Cl 2 N 9 O 24 , average mass 1449.3 amu). The standard nomenclature is used for the peptide backbone as well as for oligosaccharide fragmentation, and hence for the main fragments observed after photoabsorption. Arrows indicate on which side of the cleaved bond the charge is located. The groups #1 and 2 containing phenyl rings are encircled.

Figure 3 .Scheme 1 .

 31 Figure 3. Zoom into the region of the mass spectra of [V+2H] 2+ (see Fig. 2) where the internal fragments appear. Each of them is indicated by a letter above the corresponding group of peaks. Vertical bars correspond to the m/z of each singly-charged fragment shown in Scheme 1 with the maximum number of H atoms (leaving the bond orders unchanged), and without any H atom left. The structure of these fragments is given in Scheme 1. Scheme 1. Proposed structure corresponding to the internal fragments of [V+2H] 2+ (see Fig. 3). The bond cleavages responsible for their formation are highlighted.

Figure 4 .

 4 Figure 4. Top: mass spectra of the [V+R+2H] 2+ complex after absorption of one photon of energy between 100 and 531.5 eV. The position of the precursor ion (m/z 911.6) is represented by a purple line. The usual nomenclature is used for the peptide backbone as well as for oligosaccharide fragmentation and hence for the main fragments observed after photoabsorption (cf. Fig.1). Internal fragments of vancomycin formed by at least two bond cleavages are indicated. Bottom: relative yield of the different relaxation channels after single photoabsorption as a function of photon energy, normalized by the detector efficiency and the total yield of photoinduced cations.

Figure 5 .

 5 Figure 5. Zoom in the m/z 158-280 region of the mass spectra of vancomycin in different environments, after absorption of one 401.5 eV photon. The peaks attributed to fragments of R are spotted by an asterisk. Vertical bars correspond to the m/z of each singly-charged fragment shown in Scheme 1 with the maximum number of H atoms (leaving the bond orders unchanged), and without any H atom left.

ACKNOWLEDGMENT

We thank HZB for the allocation of synchrotron radiation beamtime and T. Kachel for his support during experiments. The French "Conseil Régional de Normandie" is acknowledged for a PhD funding (#15P01339) and CNRS for a PICS grant (07390) supporting the collaboration between CIMAP and the Open University. This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 730872. AR acknowledges Fundação para a Ciência e a Tecnologia (FCT-MCTES) for subsiding him with a PhD scholarship (grant number PD/BD/114449/2016) under the Radiation Biology and Biophysics Doctoral Training Programme (RaBBiT, PD/00193/2012) organized by the Applied Molecular Biosciences Unit -UCIBIO (UIDB/04378/2020) and CEFITEC Unit (UIDB/00068/2020) from Faculdade de Ciências e Tecnologias of Universidade Nova de Lisboa. The Sir John Mason Academic Trust is also acknowledged for the financial support provided to AR for a beamtime at HZB.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

Simulated mass spectra of singly-charged internal fragments of vancomycin shown in Scheme 1; Photoabsorption mass spectra [R 2 +H] + , [V 2 +3H] 3+ and [V+R 2 +2H] 2+ ; Total yield of internal fragments of vancomycin in different environments (PDF)

AUTHOR INFORMATION Corresponding Author

* poully@ganil.fr

Author Contributions

All authors have given approval to the final version of the manuscript.

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Mass spectral signatures of complex post-translational modifications in proteins: a proof-ofprinciple based on X-ray irradiated vancomycin

Marwa Abdelmouleh, Mathieu Lalande, Johnny El Feghaly, Violaine Vizcaino, André Rebelo, Samuel Eden, Thomas Schlathölter and Jean-Christophe Poully Gas-phase X-ray photoabsorption coupled to mass spectrometry of a heavily-modified peptide provides a robust signature of its post-translational modifications.