Gas-Phase Protein Inner-Shell Spectroscopy by ... - ACS Publications

Letter pubs.acs.org/JPCL

Gas-Phase Protein Inner-Shell Spectroscopy by Coupling an Ion Trap with a Soft X-ray Beamline Aleksandar R. Milosavljević,*,† Francis Canon,‡ Christophe Nicolas,‡ Catalin Miron,‡ Laurent Nahon,‡ and Alexandre Giuliani*,‡,§ †

Institute of Physics, University of Belgrade, Pregrevica 118, 11080 Belgrade, Serbia Synchrotron SOLEIL, L’Orme des Merisiers, Saint Aubin, BP 48, 91192 Gif-sur-Yvette, France § UAR 1008 Cepia, Institut National de la Recherche Agronomique (INRA), BP 71627, 44316 Nantes Cedex 3, France ‡

S Supporting Information *

ABSTRACT: C, N, and O near-edge ion yield spectroscopy of 8+ selected electrosprayed cations of cytochrome c protein (12 kDa) has been performed by coupling a linear quadrupole ion trap with a soft X-ray beamline. The photoactivation tandem mass spectra were recorded as a function of the photon energy. Photoionization of the precursor, accompanied by CO2 loss, is the dominant relaxation process, showing high photoion stability following direct or resonant photoionization. The partial ion yields extracted from recorded mass spectra show significantly different behaviors for single and double ionization channels, which can be qualitatively explained by different Auger decay mechanisms. However, the single ionization spectra reveal characteristic structures when compared to existing near-edge X-ray absorption fine structure (NEXAFS) spectra from thin films of peptides and proteins. Therefore, the present experiment opens up new avenues for near-edge X-ray spectroscopy of macromolecules in the gas phase, overcoming the radiation damage issue or the environmental effects as due to the surface, intermolecular interactions, and solvent. SECTION: Spectroscopy, Photochemistry, and Excited States

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gas-phase spectroscopy gives the opportunity to investigate isolated targets, under well-defined conditions. In the last 5 years, a number of studies have been also reported on gas-phase inner-shell X-ray spectroscopy of relatively small biologically relevant molecules, such as amino acids and pyrimidines3 (also see refs 10−12 and references therein). In parallel, great progress has been achieved on nearedge X-ray spectroscopy of small gaseous organic molecules by use of partial ion yield spectroscopy (see refs 13 and 14 and references therein). As a valuable alternative to photoabsorption techniques based on total electron or total ion yield measurements, and similarly to partial fluorescence yields (PFYs), the partial ion yield X-ray spectroscopy allows for the monitoring of a specific electronic transition channel and is highly selective with respect to different types of resonances, such as shape resonances15 or Auger decay processes.16 Still, a drawback of all of the above-mentioned experimental methods for neutral gaseous biological molecules is the size limitation of the target because it is rather challenging to bring a large biomolecule intact into the gas phase. The conventional thermal vaporization techniques are limited only to thermally stable samples, which is not generally the case for amino acids

n recent years, there has been a large interest to study biological systems, namely, amino acids, peptides, and proteins, by using synchrotron-based spectroscopic techniques, such as near-edge X-ray absorption fine structure (NEXAFS) or related X-ray photoelectron and X-ray emission spectroscopies. By exploiting the high spectral brightness and focusing possibilities of modern synchrotron beamlines available in the soft X-ray range, the above-mentioned techniques became efficient and versatile tools to investigate the chemical and electronic structure of organic materials and nanostructures.12 Most of the X-ray spectroscopic investigations of biologically relevant molecules, such as amino acids and their polymers, have been performed on thin organic films and liquids (see refs 3−8 and references therein). The more recent studies6−8 also revealed the possibility of using X-ray absorption for mapping peptides and proteins according to their amino acid constituents. Nevertheless, despite a number of useful sets of NEXAFS data obtained on thin organic films, some drawbacks remain. One of the most important issues is the radiation damage occurring in condensed matter samples. Indeed, amino acids and biomacromolecules are highly susceptible to soft Xrays, which leads to significant chemical transformation under long and intensive photon beam exposure.89 Furthermore, although proteins are not isolated in nature, the environmental effects in the case of studying condensed crystalline or aqueous samples can drastically affect the obtained spectra,4 whereas © 2012 American Chemical Society

Received: March 16, 2012 Accepted: April 16, 2012 Published: April 16, 2012 1191

dx.doi.org/10.1021/jz300324z | J. Phys. Chem. Lett. 2012, 3, 1191−1196

The Journal of Physical Chemistry Letters

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that would thermally decompose before obtaining sufficient vapor pressure by heating methods. Even though modern techniques such as aerosol thermodesorption sources have appeared to be very promising to investigate relatively small biomolecules in vacuo,1718 no results have been reported so far for large biosystems such as proteins. The introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) techniques permits one to bring extremely large biological compounds into the gas phase.19 In particular, tandem mass spectrometry (MS2) technique, where a given selected ionic compound is isolated, activated, and mass analyzed, has become a very efficient tool to study the structure of biopolymers.20 On this basis, coupling an ion trap mass spectrometer equipped with an ESI source to a soft X-ray synchrotron beamline provides an unprecedented opportunity to perform efficient inner-shell spectroscopy of a charge-selected gaseous biomacromolecular ion. The proof-of-principle demonstration of coupling a Fourier transform ion cyclotron resonance (FTICR) ion trap with a soft X-ray beamline was given in 2008.21 Recently, McCullough et al. reported a novel setup that uses a digital ion trap mass spectrometer for probing the structure of biomacromolecules by gas-phase small-angle X-ray scattering.22 More recently, a successful use of synchrotron radiation for VUV photoionization spectroscopy of electrosprayed biological ions stored in an ion trap has been reported.23−25 The present Letter reports on novel inner-shell measurements, performed by coupling a linear ion trap mass spectrometer, equipped with an ESI source, with the PLEIADES soft X-ray beamline at the SOLEIL synchrotron radiation facility. The electrosprayed cytochrome c ions are injected, mass selected, stored in the trap, and irradiated during a well-defined period (see Figure 1 and the Experimental

Figure 2. Tandem ESI/photoionization mass spectrum of 8+ (m/z 1546) ions of cytochrome c protein from equine heart, obtained after 600 ms of irradiation at a photon energy of 292 eV. The inset shows a zoom in of the mass regions around 9+ (m/z 1374) and 10+ (m/z 1237) charge states, normalized to the same dominant peak intensity. The arrows mark the peaks’ separations of about m/z 4.9 ± 0.1 and 4.5 ± 0.3 for the 9+ and 10+ channels, respectively, corresponding to 44 Da, tentatively assigned to CO2 loss.

ionization, M8+ + hν → M10+ + 2e−. A zoom in of the m/z 1374 and 1237 regions shows one more intense peak separated by about 44 Da (inset in Figure 2), which is likely to correspond to the CO2 loss. Therefore, the inner-shell protein photoionization is accompanied by an intense loss of carbon dioxide, which has already been observed and discussed in our most recent study of VUV ionization of cytochrome c23 and in previous investigations of electron photodetachment from gasphase deprotonated peptides.26 In the present case, the CO2 loss is significant for both single and double ionization and across all probed energy regions. All of the other ionic species have abundances lower than 0.1% and will not be discussed here. Noteworthy is the weak feature at about m/z 1767 (M7+), which can be formed through the capture of electrons (M8+ + e− → M7+) emitted by photoionization of helium bath gas (He + hν → He+ + e−). However, this influence of the He bath in the trap is obviously not significant and does not affect the present study on inner-shell protein spectroscopy because the reduced charged 7+ species have extremely low abundance. The physical processes of precursor ionization upon X-ray photon absorption, leading to the creation of a core hole, can be summarized as follows.14 Below the ionization threshold, the core electron is excited to an unoccupied bound state, and the core hole decays via resonant Auger decay or an X-ray fluorescence process (about 1% in this energy range), leading mostly to a single ionization process. Above the threshold, the dominant process is the normal Auger decay, where the core electron is ejected into the ionization continuum, followed by the relaxation of the system via an Auger electron emission, leading to a double ionization process. Therefore, monitoring the relative cross section for the production of a given ionized species (or a specific fragment ion) upon inner-shell photoexcitation of a molecule directly gives insight into a specific excitation/relaxation process. To obtain the near-edge X-ray ion yield spectra of the 8+ charge state precursor, tandem mass spectra were recorded as a function of the photon energy, in small steps of 0.2 eV, in the

Figure 1. Principle of the experimental method.

Section for more details). To the best of our knowledge, this is the first report on soft X-ray near-edge spectroscopy of a protein isolated in the gas phase, which opens up promising opportunities for near-edge X-ray spectroscopy of biological macromolecules. Figure 2 shows a typical MS2 spectrum of electrosprayed cytochrome c protein, recorded after 600 ms of irradiation of the 8+ charge state isolated precursor (M8+, m/z 1546) at the photon energy of 292 eV. Clearly, the photoionization channels of the precursor represent the major relaxation pathways. The peak at m/z 1374 corresponds to single ionizatio,: M8+ + hν → M9+ + e−, whereas the one at m/z 1237 corresponds to double 1192



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M9+ cation yield, corresponding to the single ionization, exhibits a rich spectroscopic structure below the thresholds. Moreover, complementary to the increase of the M10+ yield, a significant decrease of the single ionization is seen above the thresholds. It should be noted that a similar difference between singly and doubly charged cation yields has been previously observed in partial ion yield spectroscopy of small neutral molecules.1314 Following the latter studies, the current observed differences between the single and double ionization of the protein precursor can be tentatively explained by distinct Auger decay mechanisms. Below the threshold, the resonant Auger decay mechanism is operative; therefore, the photoexcitation to a bound molecular state leads predominantly to the single ionization. The threshold jump in the production of the doubly ionized precursor (consequently, a decrease in the single ionization efficiency) can be thus related to the transition from the resonant Auger decay to the normal Auger decay taking place above the threshold. For a comparison, previously reported3 ionization thresholds for glycyl−glycine dipeptide are shown in Figure 3 as well. The latter data for C 1s ionization matches very well the present results, while the lowest reported ionization potentials of glycyl−glycine for N and O K-shells are somewhat below the present double ionization onsets. Note also that multiple thresholds are not observed in the present M10+ partial ion yield. It should be noted that M10+ cations can be also produced below the thresholds and even below the first resonances, as can be observed especially for N and O edges (Figure 3b and c). Usually, this kind of behavior is not observed for small molecular systems, very likely because of the fragmentation of the doubly charged species additionally stimulated by Coulomb repulsion. However, in the present case, the 10+ cation is astonishingly stable. This observation might have important consequences for radiobiology and radiation damage of large systems by X-rays. In particular, the classical bottom-up approach, which consists of studying the so-called life building blocks, is questioned by the different stabilities of proteins and isolated amino acids. This exceptional stability of the protein could not be inferred from the photochemical properties of the individual amino acids. Thus, the flat region of the relative 10+ cation yield, below the N and O edges, might come from the normal Auger decay events corresponding to the lower-lying Kedges. In contrast, the weak structures that are observed in the M10+ yields on the top of these monotonous pre-edge curves and at the same photon energies as those for the M9+ yields (resonances D and F) could reflect the population of spectatortype resonant Auger decay, which will further relax via a cascade or second-step Auger process toward double ionization channels. The C 1s single ionization spectrum is presented in Figure 3a. It should be noted that a prominent dip at about 284.5 eV is most probably due to a normalization of weak background signal to a very steep photon flux curve in this energy region. The peak A at about 285.5 eV is assigned to the C 1s → π* transitions associated with the aromatic rings of amino acid residues. This feature has been reported in previous NEXAFS spectra of protein solid films as a signature of aromatic amino acids (Phe, Tyr, Trp).36−8 Stewart-Ornstein et al.6 have tabulated the energies and assignments of NEXAFS spectral features for several peptides and small proteins. The corresponding features have been assigned (final orbital) to π*CC(aromatic)(ν = 0) and π*CC(aromatic)(ν = 1) of the Trp residue, with values in the range of 285.13−285.15 and

vicinity of the C, N, and O K-edges. The intensity of the peaks corresponding to single or double ionization processes was normalized to both the total ion current and photon flux and plotted against the photon energy. The resulting spectra are shown in Figure 3. The results reveal a fundamentally different energy dependence of the ion yields corresponding to the M9+ (circles, single ionization) and M10+ (diamonds, double ionization) channels. In the latter case, the photoionization curves are basically flat below the threshold. In contrast, the

Figure 3. (a−c) Single (M9+, 9+ charge state, m/z 1373−1375) and double (M10+, 10+ charge state, m/z 1236−1237.5) C, N, and O Kedge photoionization yields of the 8+ charge state precursor (M8+) of equine cytochrome c protein. Vertical lines mark tabulated K-shell ionization thresholds for glycyl−glycine.3 (d) Top panel: C K-edge total photoionization ion yield (TIY, m/z 1000−1400). (d) Bottom panel: Comparison of M9+ and M10+ C K-edge photoionization yields with corresponding [M−CO2]9+ (m/z 1368−1370) and [M−CO2]10+ (m/z 1231.5−1233) yields (see Figure 2) normalized to the same intensity at higher photon energies. 1193



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peak E centered at about 406 eV is dominantly associated with σ* transitions (the resonance σ*C−N was reported in the range of 406.2−406.6 eV).6 Similarly, the dominant feature F in the O 1s spectrum, at about 531.4 eV, corresponds to O 1s→π*amide transitions. Gordon et al.3 found the value of 531.6 eV for fibrinogen and pointed out that the position of the dominant peak shifts to lower energies when going from small polypeptides to the protein. Having in mind that the present inner-shell spectroscopy is actually performed on ionic precursors in the gas phase (protonated protein), an important issue to address is a possible influence of the protonation to the recorded spectra. It has been suggested that the charge state and solvation environment could strongly influence the NEXAFS spectra.4 Because the addition of a proton changes the electric field symmetry in the vicinity of terminal nitrogen or oxygen, a change of the pH would result in a significant change of the NEXAFS spectrum. Messer et al.4 performed the first systematic NEXAFS study of an amino acid in an aqueous solution by exploiting the technology of liquid microjets. They indeed concluded that all three, carbon, nitrogen, and oxygen, K-edge spectra exhibited a dependence on solution pH, and the most significant changes were seen for the N K-edge. Recently, a similar study on aqueous adenosine triphosphate also revealed dramatic pH dependence for both carbon and nitrogen spectra, which were rationalized based on symmetry changes in the adenine ring.27 However, a basic difference between the previous spectroscopy of relatively small aqueous biomolecules and the case of a large gas-phase protonated macromolecule must be stressed here. First, in the present case, no water molecules are present around the target species, which could influence the spectra (e.g., through hydrogen bond formation). Second, the local electric field is disturbed at only a few protonated sites over a large molecular system, which might have a restricted and negligible influence on the final spectra. Indeed, the positions of spectral features measured for the 8+ precursor in the present study accurately correspond to previous results on condensed proteins. In conclusion, we have demonstrated a novel possibility to efficiently perform inner-shell spectroscopy of large electrosprayed gaseous macromolecular ions by coupling a linear quadrupole ion trap with a soft X-ray synchrotron beamline. Therefore, intact compounds of practically unlimited sizes and various charge states can be isolated in the vacuum by use of an ESI source and studied by means of near-K-edge ion yield spectroscopy. Single and double ionization of the 8+ charge state precursor of cytochrome c protein has been investigated in detail. The obtained single ionization yield spectra appeared to be generally similar to previous solid film NEXAFS results (except the resonant peak’s intensity ratios in the C 1s spectrum), allowing tentative assignments of spectral features. Thus, the present technique opens up new opportunities for near-edge X-ray spectroscopy of biological macromolecules in the gas phase as a complementary technique to NEXAFS performed on thin organic films and liquids while overcoming the serious radiation damage issue as well as the surface, intermolecular, and solvent effects. Furthermore, the relaxation processes in macromolecular systems could be studied by combining the possibility to probe a wide range of selected poly(de)protonated ionic states with the use of the highly selective partial ion yield technique. Finally, a striking observation is the apparent stability of the large photoions arising from direct photoionization or Auger processes. Indeed,

285.52−285.58 eV, respectively (indocilin, sub6, albumin, fibrinogen). Note also that a second closely lying peak assigned to C 1s(C−R) → π*CC of the Trp residue6 at about 286.05 eV can be seen as a shoulder in the present spectrum at 286.1 eV. Therefore, the fingerprint of the aromatic amino acids can be well-resolved by using the present method, the resonances correlating very closely to solid film NEXAFS studies. The small but distinguishable structure at 287.0 eV can be assigned to σ*C−H resonance, tabulated previously at the same energy.6 The next strong peak B in the present spectrum, at about 288.3 eV, can be assigned to the C 1s→π*amide transition.6 The energy position is in perfect agreement with the previously reported value of 288.2 eV for albumin and fibrinogen proteins,6 as well as that for very large protein powder samples (lysozime, ovalbumin, collagen).7 The broad peak C at about 293 eV corresponds to the overlap of various transitions, mainly associated with the σ* resonance (C−C, C−N, C−O, ...). This part of the spectrum is related to valence and Rydberg transitions of amino acid residues, which merge into a broad band, as expected for a large polypeptide. What is interesting, however, when the present C 1s spectrum of gas-phase cytochrome c is compared to the majority of previous X-ray absorption spectra of condensed proteins, is a rather low intensity of the π*amide peak (B) relative to both the aromatic peak (A) and the broad σ* peak (C). An explanation based on a selective ejection of CO2 at the 288.3 eV resonant energy should be ruled out because corresponding ion yields appear to be very similar (see Figure 3d, bottom panel); actually, the CO2 loss seems to be reduced at the π*amide resonance. Moreover, the shape of the total photoionization ion yield curve (TIY, Figure 3d, top panel), which should correspond to the absorption spectrum, differs from existing NEXAFS spectra for condensed proteins.67 A definite explanation of this effect cannot be given before further research on more samples is conducted; however, one can distinguish two possible sources of the discrepancy, an experimental influence and/or a sample=related effect (specific hemoprotein, multiply protonated, gas-phase sample). In the former case, a possible explanation could be the influence of the photon flux, which is highly energy-sensitive at around 288 eV (the so-called “carbon dip” due to carbon contamination of the beamline). Note that N and O K-edge spectra appear to correspond well in shape to previous NEXAFS results. On the other hand, it should be also considered that previous results67 did show C K-edge π*amide resonance to be rather sensitive to the protein sequence, which is different in the case of the present cytochrome c protein (which also contains heme). Finally, until further investigation is conducted, an influence of a high precursor charge state to the spectral shape is not known. The N 1s and O 1s single ionization spectra (Figure 3b and c, respectively) are similar to previous NEXAFS results reported for various proteins and polypeptides.6−8 In general, the distinctive spectral features are broadened and smeared-out in the case of a large macromolecule, as suggested earlier.8 The dominant peak D found in the N 1s spectrum at 401.4 eV corresponds to the N 1s→π*amide [N 1s→π*CO(CONH)] transition. A similar energy was reported by Gordon et al.3 for polypeptides and fibrinogen, while Stewart-Ornstein et al.6 reported 401.2 eV (indocilin, sub6, albumin, fibrinogen). Note also a relatively well-resolved feature in the present spectrum at about 403.2 eV, which could be tentatively assigned to π*CN that was previously found to be at about 403 eV.6 The broad 1194



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the major fragmentation channel appears to be CO2 losses and does not involve peptidic backbone cleavages.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information about near-edge X-ray ion yield spectroscopy of selected electrosprayed cations of PPG. This material is available free of charge via the Internet at http:// pubs.acs.org.

EXPERIMENTAL SECTION

A general scheme of coupling the linear quadrupole ion trap to a synchrotron beamline has been described in recent publications.2325 The experimental setup is based on a commercial linear quadrupole ion trap mass spectrometer (Thermo Finnigan LTQ XL) equipped with an ESI source. The electrosprayed ions are introduced from the front side into the trap, while the soft X-ray photon beam is sent into the trap from the back side. The irradiation time (600 ms in the present case) of mass-selected precursors is regulated by a special photon shutter28 triggered by the spectrometer. The measurement process (changing the photon energy and acquisition of tandem mass spectra) is automatic and synchronized by homemade software. The setup also includes a differential pumping stage to accommodate the pressure difference between the beamline (10−9 mbar) and the LTQ (10−5 mbar of He in the main chamber). The setup is connected to the soft X-ray beamline PLEIADES29 of the SOLEIL storage ring in St. Aubin (France). The photon beam is produced by a quasi-periodic APPLE II type of undulator (HU80), followed by a modified Petersen plane grating monochromator with varied line spacing and varied groove depth gratings. The latter allow one to keep the monochromator diffraction efficiency optimal for arbitrarily chosen photon energies within the operating range.30 For the present experiment, 400 lines mm−1 (at C and N edges) and 1600 lines mm−1 (at the O edge) gratings were used, which provide high photon flux on the order of (1−2) × 1012 photons s−1/0.1% bandwidth in the used energy range.31 For the 400 lines mm−1 grating, the energy resolution used (full with at halfmaximum, fwhm) was about 250−300 meV in the energy range of 280−300 eV (C edge) and about 430−460 meV in the energy range of 395−415 eV (N edge). For the 1600 lines mm−1 grating, the energy resolution used was about 260−400 meV in the energy range of 525−545 eV. The photon energy was calibrated according to the Ar(2p3/2−14s), N 1s→π* in N2, O 1s→π*, and O 1s→3sσ* in O2 resonances.32−34 The absolute accuracy of the energy calibration was estimated to be 50 meV for the C and N edges and 100 meV for the O edge. Multiply protonated cytochrome c from equine heart (Sigma Aldrich) molecules was generated by the ESI source from a water/acetonitrile (75:25) solution at 10 μM. It should be noted that the present mass resolution is not sufficient to establish unambiguously the charge state of the heme iron; consequently, the isolated precursor ion in an n+ charged state could be assigned to either [M + nH]n+ or [M + (n − 1)H]n+ for either the Fe(II) or Fe(III) heme iron oxidation state, respectively.23 In order to test the methodology, the inner-shell spectroscopy of the polypropylene glycol polymer (PPG), which contains neither nitrogen atoms nor double bonds, has been performed under the same conditions. The results are shown in the Supporting Information. As expected, there is a clear difference between results obtained for cytochrome c and those for the PPG molecules, which confirms the high efficiency of the experimental method to accurately probe the trapped selected ions, with a negligible influence of indirect effects.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (A.R.M.); giuliani@synchrotron-soleil. fr (A.G.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Data collection was performed on the PLEIADES beamline at the SOLEIL Synchrotron (Proposal Number 20100752). This work is supported by the Agence Nationale de la Recherche Scientifique (Project #BLAN08-1_348053). A.R.M. acknowledges support for a short-term scientific mission to SOLEIL from the Institute Français de Serbie through the EGIDE program, as well as support by the Ministry of Education and Science of the Republic of Serbia (Project No. 171020). We are grateful to E. Robert for technical assistance and the general SOLEIL staff for running the overall facility.

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REFERENCES

(1) Hähner, G. Near Edge X-ray Absorption Fine Structure Spectroscopy as a Tool to Probe Electronic and Structural Properties of Thin Organic Films and Liquids. Chem. Soc. Rev. 2006, 35, 1244− 1255. (2) Aziz, E. F. X-ray Spectroscopies Revealing the Structure and Dynamics of Metalloprotein Active Centers. J. Phys. Chem. Lett. 2011, 2, 320−326. (3) Gordon, M. L.; Cooper, G.; Morin, C.; Araki, T.; Turci, C. C.; Kaznatcheev, K.; Hitchcock, A. P. Inner-Shell Excitation Spectroscopy of the Peptide Bond: Comparison of the C 1s, N 1s, and O 1s Spectra of Glycine, Glycyl−Glycine, and Glycyl−Glycyl−Glycine. J. Phys. Chem. A 2003, 107, 6144−6159. (4) Messer, B. M.; Cappa, C. D.; Smith, J. D.; Wilson, K. R.; Gilles, M. K.; Cohen, R. C.; Saykally, R. J. pH Dependence of the Electronic Structure of Glycine. J. Phys. Chem. B 2005, 109, 5375−5382. (5) Zubavichus, Y.; Shaporenko, A.; Grunze, M.; Zharnikov, M. NEXAFS Spectroscopy of Homopolypeptides at All Relevant Absorption Edges: Polyisoleucine, Polytyrosine, and Polyhistidine. J. Phys. Chem. B 2007, 111, 9803−9807. (6) Stewart-Ornstein, J.; Hitchcock, A. P.; Hernández, C. D.; Henklein, P.; Overhage, J.; Hilpert, K.; Hale, J. D.; Hancock, R. E. W. Using Intrinsic X-ray Absorption Spectral Differences to Identify and Map Peptides and Proteins. J. Phys. Chem. B 2007, 111, 7691−7699. (7) Zubavichus, Y.; Shaporenko, A.; Grunze, M.; Zharnikov, M. Is Xray Absorption Spectroscopy Sensitive to the Amino Acid Composition of Functional Proteins? J. Phys. Chem. B 2008, 112, 4478−4480. (8) Zubavichus, Y.; Shaporenko, A.; Grunze, M.; Zharnikov, M. NEXAFS Spectroscopy of Biological Molecules: From Amino Acids to Functional Proteins. Nucl. Instrum. Methods Phys. Res., Sect. A 2009, 603, 111−114. (9) Kade, A.; Kummer, K.; Vyalikh, D. V.; Danzenbächer, S.; Blüher, A.; Mertig, M.; Lanzara, A.; Scholl, A.; Doran, A.; Molodtsov, S. L. Xray Damage in Protein−Metal Hybrid Structures: A Photoemission Electron Microscopy Study. J. Phys. Chem. B 2010, 114, 8284−8289. (10) Marinho, R. R. T.; Lago, A. F.; Homem, M. G. P.; Coutinho, L. H.; de Souza, G. G. B.; de Brito, A. N. Gas Phase Photoabsorption and 1195



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Synchrotron Radiation Quadrupole Ion Trap Tandem Mass Spectrometry. Nucl. Instrum. Methods Phys. Res., Sect. B 2011, 279, 34−36. (29) PLEIADES beamline website. http://www.synchrotron-soleil.fr/ Recherche/LignesLumiere/PLEIADES (accessed March 15, 2012). (30) Sö derströ m, J.; Lindblad, A.; Grum-Grzhimailo, A. N.; Travnikova, O.; Nicolas, C.; Svensson, S.; Miron, C. Angle-Resolved Electron Spectroscopy of the Resonant Auger Decay in Xenon with meV Energy Resolution. New J. Phys. 2011, 13, 073014/1−073014/13. (31) Miron, C.; Nicolas, C.; Travnikova, O.; Morin, P.; Sun, Y.; Gelmukhanov, F.; Kosugi, N.; Kimberg, V. Imaging Molecular Potentials Using Ultrahigh-Resolution Resonant Photoemission. Nat. Phys. 2012, 8, 135−138. (32) Sairanen, O.-P.; Kivimäki, A.; Nõmmiste, E.; Aksela, H.; Aksela, S. High-Resolution Pre-Edge Structure in the Inner-Shell Ionization Threshold Region of Rare Gases Xe, Kr, and Ar. Phys. Rev. A 1996, 54, 2834−2839. (33) Piancastelli, M. N.; Kivimäki, A.; Kempgens, B.; Neeb, M.; Maier, K.; Hergenhahna, U.; Rüdela, A.; Bradshawa, A. M. Electron Decay Following the N 1s→π* Excitation in N2 Studied Under Resonant Raman Conditions. J. Electon Spectrosc. Relat. Phenom. 1999, 98−99, 111−120. (34) Tanaka, T.; Feifel, R.; Kitajima, M.; Tanaka, H.; Sorensen, S. L.; Sankari, R.; De Fanis, A.; Piancastelli, M.-N.; Karlsson, L.; Ueda, K. Symmetry-Resolved X-ray Absorption Fine Structure and Resonant Auger-Spectator-Electron Decay Study of O 1s→Rydberg Resonances in O2. Phys. Rev. A 2008, 78, 022516/1−022516/11.

Mass Spectra of L-Alanine and L-Proline in the Soft X-ray Region. Chem. Phys. 2006, 324, 420−424. (11) Lessard, R.; Cuny, J.; Cooper, G.; Hitchcock, A. P. Inner-Shell Excitation of Gas Phase Carbonates and a,c-Dicarbonyl Compounds. Chem. Phys. 2007, 331, 289−303. (12) Bolognesi, P.; O’Keeffe, P.; Ovcharenko, Y.; Coreno, M.; Avaldi, L.; Feyer, V.; Plekan, O.; Prince, K. C.; Zhang, W.; Carravetta, V. Pyrimidine and Halogenated Pyrimidines Near Edge X-ray Absorption Fine Structure Spectra at C and N K-Edges: Experiment and Theory. J. Chem. Phys. 2010, 133, 034302−034302. (13) Guillemin, R.; Stolte, W. C.; Lindle, D. W. Fragmentation of Formic Acid Following Photoexcitation Around the Carbon K Edge. J. Phys. B: At. Mol. Opt. Phys. 2009, 42, 125101/1−125101/7. (14) Guillemin, R.; Stolte, W. C.; Piancastelli, M. N.; Lindle, D. W. Jahn-Teller Coupling and Fragmentation After Core−Shell Excitation in CF4 Investigated by Partial-Ion-Yield Spectroscopy. Phys. Rev. A 2010, 82, 043427/1−043427/7. (15) Piancastelli, M. N. The Neverending Story of Shape Resonances. J. Electron Spectrosc. Relat. Phenom. 1999, 100, 167−190. (16) Sorensen, S.; Miron, C.; Feifel, R.; Miancastelli, M. N.; Björneholm, O.; Svensson, S. The Influence of the σ Resonance on the Auger Decay of Core-Ionized Molecular Nitrogen. Chem. Phys. Lett. 2008, 456, 1−6. (17) Wilson, K. R.; Jimenez-Cruz, M.; Nicolas, C.; Belau, L.; Leone, S. R.; Ahmed, M. Thermal Vaporization of Biological Nanoparticles: Fragment-Free Vacuum Ultraviolet Photoionization Mass Spectra of Tryptophan, Phenylalanine−Glycine−Glycine, and β-Carotene. J. Phys. Chem. A 2006, 110, 2106−2113. (18) Gaie-Levrel, F.; Garcia, G. A.; Schwell, M.; Nahon, L. VUV State-Selected Photoionization of Thermally-Desorbed Biomolecules by Coupling an Aerosol Source to an Imaging Photoelectron/ Photoion Coincidence Spectrometer: Case of the Amino Acids Tryptophan and Phenylalanine. Phys. Chem. Chem. Phys. 2011, 13, 7024−7036. (19) Baker, M. Mass Spectrometry for Biologists. Nat. Methods 2010, 7, 157−161. (20) Aebersold, R.; Mann, M. Mass Spectrometry-Based Proteomics. Nature 2003, 422, 198−207. (21) Thissen, R.; Bizau, J. M.; Blancard, C.; Coreno, M.; Dehon, C.; Franceschi, P.; Giuliani, A.; Lemaire, J.; Nicolas, C. Photoionization Cross Section of Xe+ Ion in the Pure 5p5 2P3/2 Ground Level. Phys. Rev. Lett. 2008, 100, 223001/1−223001/4. (22) McCullough, B. J.; Entwistle, A.; Konishi, I.; Buffey, S.; Hasnain, S. S.; Brancia, F. L.; Grossmann, J. G.; Gaskell, S. J. Digital Ion Trap Mass Spectrometer for Probing the Structure of Biological Macromolecules by Gas Phase X-ray Scattering. Anal. Chem. 2009, 81, 3392− 3397. (23) Milosavljević, A. R.; Nicolas, C.; Lemaire, J.; Déhon, C.; Thissen, R.; Bizau, J.-M.; Réfrégiers, M.; Nahon, L.; Giuliani, A. Photoionization of a Protein Isolated In Vacuo. Phys. Chem. Chem. Phys. 2011, 13, 15432−15436. (24) Bari, S.; Gonzalez-Magana, O.; Reitsma, G.; Werner, J.; Schippers, S.; Hoekstra, R.; Schlatholter, T. Photodissociation of Protonated Leucine-Enkephalin in the VUV Range of 8−40 eV. J. Chem. Phys. 2011, 134, 024314−024319. (25) Milosavljević, A. R.; Nicolas, C.; Gil, J.-F.; Canon, F.; Réfrégiers, M.; Nahon, L.; Giuliani, A. VUV Synchrotron Radiation: a New Activation Technique for Tandem Mass Spectrometry. J. Synchrotron Radiat. 2012, 19, 174−178. (26) Joly, L.; Antoine, R.; Broyer, M.; Lemoine, J.; Dugourd, P. Electron Photodetachment from Gas Phase Peptide Dianions. Relation with Optical Absorption Properties. J. Phys. Chem. A 2008, 112, 898− 903. (27) Kelly, D. N.; Schwartz, C. P.; Uejio, J. S.; Duffin, A. M.; England, A. H.; Saykally, R. J. Near Edge X-ray Absorption Fine Structure Spectroscopy of Aqueous Adenosine Triphosphate at the Carbon and Nitrogen K-Edges. J. Chem. Phys. 2010, 133, 101103−101106. (28) Milosavljević, A. R.; Nicolas, C.; Gil, J.-F.; Canon, F.; Réfrégiers, M.; Nahon, L.; Giuliani, A. Fast In Vacuo Photon Shutter for 1196


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