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Silver has been used for its antimicrobial properties to fight infection for thousands of years. Unfortunately, some Gram-negative bacteria have developed silver resistance causing the death of patients in a burn unit. The genes responsible for silver resistance have been designated as the sil operon. Among the proteins of the sil operon, SilE has been shown to play a key role in bacterial silver resistance. Based on the limited information available, it has been depicted as an intrinsically disordered protein that folds into helices upon silver ion binding. Herein, this work demonstrates that SilE is composed of 4 clearly identified helical segments in the presence of several silver ions. The combination of analytical and biophysical techniques (NMR spectroscopy, CD, SAXS, HRMS, CE-ICP-MS, and IM-MS) reveals that SilE harbors four strong silver binding sites among the eight sites available. We have also further evidenced that SilE does not adopt a globular structure but rather samples a large conformational space from elongated to more compact structures. This particular structural organization facilitates silver binding through much higher accessibility of the involved His and Met residues. These valuable results will advance our current understanding of the role of SilE in the silver efflux pump complex mechanism and will help in the future rational design of inhibitors to fight bacterial silver resistance.

Chemistry - a European journal 27(48), 12371 - 12379 (2021). doi:10.1002/chem.202101515, We investigated the photoionization and fragmentation of isolated metal protoporphyrin IX cations (MPPIX+ with M=Fe, Co, Zn) by means of vacuum-ultraviolet (VUV) action spectroscopy in the energy range of 8.5���35 eV. Experiments were carried out in the gas phase by interfacing an electrospray ionization tandem mass spectrometer with a synchrotron beamline. The mass spectra and partial ion yields show that photoexcitation of the precursor ions predominantly leads to .CH$_2$COOH radical side-chain losses of the macrocycle with additional methyl radical (.CH$_3$) side-chain losses. Ionization, in contrast, leads to the formation of the intact ionized precursor and various doubly charged fragments which are mostly due to side-chain cleavages. Although statistical fragmentation dominates, we found evidence for non-statistical processes such as new fragments involving for example single and double H2O losses, indicating that different relaxation mechanisms are at play upon photoionization compared to photoexcitation. The measured ionization energies were 9.6��0.2 eV, 9.4��0.2 eV and 9.6��0.2 eV for FePPIX$^+$, CoPPIX$^+$ and ZnPPIX$^+$, respectively., Published by Wiley-VCH, Weinheim

International audience; Cysteine (Cys) is prone to diverse post-translational modifications in proteins, including oxidation into sulfenic acid (Cys-SOH) by reactive oxygen species generated under oxidative stress. Detection of low concentrated and metastable Cys-SOH within complex biological matrices is challenging due to the dynamic concentration range of proteins in the samples. Herein, visible laser-induced dissociation (LID) implemented in a mass spectrometer was used for streamlining the detection of Cys oxidized proteins thanks to proper derivatization of Cys-SOH with a chromophore tag functionalized with a cyclohexanedione group. Once grafted, peptides undergo a high fragmentation yield under LID, leading concomitantly to informative backbone ions and to a chromophore reporter ion. 79 % of the Cys-containing tryptic peptides deriving from human serum albumin and serotransferrin tracked by Parallel Reaction Monitoring (PRM) were detected as targets subjected to oxidation. These candidates, as well as Cys-containing peptides predicted by in silico trypsin digestion of 5 other human plasma proteins, were then tracked in real plasma samples to pinpoint the endogenous Cys-SOH subpopulation. Most of the targeted peptides were detected in all plasma samples by LID-PRM, with significant differences in their relative amounts. By eliminating the signal of interfering co-eluted compounds, LID-PRM surpasses conventional HCD-PRM in detecting grafted Cys-SOH containing peptides and allows now to foresee clinical applications in large human cohorts.

International audience; Cysteine (Cys) is subject to a variety of reversible post-translational modifications such as formation of sulfenic acid (Cys-SOH). If this modification is often involved in normal biological activities, it can also be the result of oxidative damage. Indeed, oxidative stress yields abnormal cysteine oxidations that affect protein function and structure and can lead to neurodegenerative diseases. In a context of population ageing, validation of novel biomarkers for detection of neurodegenerative diseases is important. However, Cys-SOH proteins investigation in large human cohorts is challenging due to their low abundance and lability under endogenous conditions. To improve the detection specificity towards the oxidized protein subpopulation, we developed a method that makes use of a mass spectrometer coupled with visible laser induced dissociation (LID) to add a stringent optical specificity to the mass selectivity. Since peptides do not naturally absorb in the visible range, this approach relies on the proper chemical derivatization of Cys-SOH with a chromophore functionalized with a cyclohexanedione. To compensate for the significant variability in total protein expression within the samples and any experimental bias, a normalizing strategy using free thiol (Cys-SH) cysteine peptides derivatized with a maleimide chromophore as internal references was used. Thanks to the differential tagging, oxidative ratios were then obtained for 69 Cys-containing peptides from 19 proteins tracked by parallel reaction monitoring (PRM) LID, in a cohort of 49 human plasma samples from Alzheimer disease (AD) patients. A statistical analysis indicated that, for the proteins monitored, the Cys oxidative ratio does not correlate with the diagnosis of AD. Nevertheless, the PRM-LID method allows the unbiased, sensitive and robust relative quantification of Cys oxidation within cohorts of samples.

Atomically precise, ligand-protected gold nanoclusters (AuNCs) attract considerable attention as contrast agents in the biosensing field. However, the control of their optical properties and functionalization of surface ligands remain challenging. Here we report a strategy to tailor AuNCs for the precise detection of protein carbonylation-a causal biomarker of ageing. We produce Au(15)SG(13) (SG for glutathione) with atomic precision and functionalize it with a thiolated aminooxy moiety to impart protein carbonyl-binding properties. Mass spectrometry and molecular modelling reveal the key structural features of Au(15)SG(12)-Aminooxy and its reactivity towards carbonyls. Finally, we demonstrate that Au(15)SG(12)-Aminooxy detects protein carbonylation in gel-based 1D electrophoresis by one- and two-photon excited fluorescence. Importantly, to our knowledge, this is the first application of an AuNC that detects a post-translational modification as a nonlinear optical probe. The significance of post-translational modifications in life sciences may open avenues for the use of Au(15)SG(13) and other nanoclusters as contrast agents with tailored surface functionalization and optical properties. Atomically precise gold nanoclusters hold promise as non-linear optical probes for biological imaging. Here functionalized luminescent gold nanoclusters bind free carbonyls via oxime bond formation, allowing detection of carbonylated proteins via gel electrophoresis and fluorescence imaging.

Cysteine (Cys) is prone to diverse post-translational modifications in proteins, including oxidation into sulfenic acid (Cys-SOH) by reactive oxygen species generated under oxidative stress. Detection of low-concentration and metastable Cys-SOH within complex biological matrices is challenging due to the dynamic concentration range of proteins in the samples. Herein, visible laser-induced dissociation (LID) implemented in a mass spectrometer was used for streamlining the detection of Cys oxidized proteins owing to proper derivatization of Cys-SOH with a chromophore tag functionalized with a cyclohexanedione group. Once grafted, peptides undergo a high fragmentation yield under LID, leading concomitantly to informative backbone ions and to a chromophore reporter ion. Seventy-nine percent of the Cys-containing tryptic peptides derived from human serum albumin and serotransferrin tracked by parallel reaction monitoring (PRM) were detected as targets subjected to oxidation. These candidates as well as Cys-containing peptides predicted by

Abstract VUV action spectroscopy has recently gained interest for the study of peptides and proteins. However, numerous aspects of the fundamental processes involved in the photodissociation are yet to be understood. It can, for example, be expected that sulfur-containing amino-acid residues have a significant impact on the dissociation processes following photoionization because of their potential involvement in the transport of electron holes in proteins. In order to investigate the influence of the sulfur-containing methionine residue on the VUV photodissociation of a small peptide a VUV action spectroscopy study of gas-phase protonated methionine-enkephalin and leucine-enkephalin in the photon energy range of 6–35 eV was performed. The results show that upon non-ionizing photoexcitation, the fragmentation patterns of the two peptides are nearly identical, whereas significant differences were observed upon photoionization. The differences between the fragment yields and the identified specific dissociation channels for methionine-enkephalin could be explained by the high electron hole affinity of sulfur, which efficiently directs the radical to the methionine side chain. Additionally, for both peptides the presence of the intact photoionized precursor ions was confirmed by their isotopic patterns and the stability of these species could be evaluated. Graphic abstract

The nature of salts introduced in the chromatographic mobile phase to promote on-line electrospray ionization was shown to be a key parameter in the optimization of LCCC-MS couplings, both using non-polar (Si-C18) and polar (Si-NH2) stationary phases. The critical conditions of poly(ethylene glycol), which reflect a compensation process between exclusion and interaction effects, were strongly modified when changing the size of the cation in the eluent. This phenomenon could be attributed to interactions between the cation and water molecules from the mobile phase, in the case of the non-polar stationary phase, giving rise to a salting out effect due to a lowered solvent quality of the eluent; or between the cation and amino-modified silanols of the polar stationary phase, inducing a decrease of the surface adsorptivity. Accordingly, increasing the size of the cation in the mobile phase caused the polymer molecules to be eluted according to the adsorption mode using non-polar adsorbent and according to the exclusion mode using polar stationary phase.

International audience; Mass spectrometry offers an arsenal of tools for diverse proteomic investigations. This perspective article reviews some of the recent developments in the field of coupling laser‐induced dissociation with mass spectrometry (LID‐MS). Strategies involving labelling with a chromophore to induce specific photo‐absorption properties are considered, with a focus on specific amino acid derivatization. Some of the opportunities and challenges of LID‐MS after targeted labelling for increasing specificity in complex sample analysis are discussed.

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International audience; A pump–probe approach was designed to determine the internal proton transfer (PT) rate in a series of poly-peptide radical cations containing both histidine and tryptophan. The proton transfer is driven by the gas-phase basicity difference between residues. The fragmentation scheme indicates that the gas-phase basicity of histidine is lower than that of radical tryptophan so that histidine is always pulling the proton away from tryptophan. However, the proton transfer requires the two basic sites to be in close proximity, which is rate limited by the peptide conformational dynamics. PT rate measurements were used to probe and explore the peptide conformational dynamics in several poly-glycines/prolines/alanines. For small and unstructured peptides, the PT rate decreases with the size, as expected from a statistical point of view in a flat conformational space. Conversely, if structured conformations are accessible, the structural flexibility of the peptide is decreased. This slows down the occurrence of conformations favorable to proton transfer. A dramatic decrease in the PT rates was observed for peptides HAnW, when n changes from 5 to 6. This is attributed to the onset of a stable helix for n = 6. No such discontinuity is observed for poly-glycines or poly-prolines. In HAnW, the gas-phase basicity and helix propensity compete for the position of the charge. Interestingly, in this competition between PT and helix formation in HA6W, the energy gain associated with helix formation is large enough to slow down the PT beyond experimental time but does not ultimately prevail over the proton preference for histidine

International audience; The nonapeptide oxytocin (OT) is used as a model sulfur-containing peptide to study the damage induced by vacuum UV (VUV) radiations. In particular, the effect of the presence (or absence in reduced OT) of oxytocin's internal disulfide bridge is evaluated in terms of photo-fragmentation yield and nature of the photo-fragments. Intact, as well as reduced, OT is studied as dianions and radical anions. Radical anions are prepared and photo-fragmented in two-color experiments (UV + VUV) in a linear ion trap. VUV photo-fragmentation patterns are analyzed and compared, and radical-induced mechanisms are proposed. The effect of VUV is principally to ionize but secondary fragmentation is also observed. This secondary fragmentation seems to be considerably enabled by the initial position of the radical on the molecule. In particular, the possibility to form a radical on free cysteines seems to increase the susceptibility to VUV fragmentation. Interestingly, disulfide bridges, which are fundamental for protein structure, could also be responsible for an increased resistance to ionizing radiations.

The authors would like to thank Diane Cala and Ruth Rimokh for providing the Escherichia coli and human HS578T cells, as well as Joelle Saulnier for her assistance in protein concentration measurements.; International audience; Thanks to comprehensive and unbiased sampling of all precursor ions, the interest to move toward bottom-up proteomic with data-independent acquisition (DIA) is continuously growing. DIA offers precision and reproducibility performances comparable to true targeted methods but has the advantage of enabling retrospective data testing with the hypothetical presence of new proteins of interest. Nonetheless, the chimeric nature of DIA MS/MS spectra inherent to concomitant transmission of a multiplicity of precursor ions makes the confident identification of peptides often challenging, even with spectral library-based extraction strategy. The introduction of specificity at the fragmentation step upon ultraviolet or visible laser-induced dissociation (LID) range targeting only the subset of cysteine-containing peptides (Cys-peptide) has been proposed as an option to streamline and reduce the search space. Here, we describe the first coupling between DIA and visible LID at 473 nm to test for the presence of Cys-peptides with a peptide-centric approach. As a test run, a spectral library was built for a pool of Cys-synthetic peptides used as surrogates of human kinases (1 peptide per protein). By extracting ion chromatograms of query standard and kinase peptides spiked at different concentration levels in an Escherichia coli proteome lysate, DIA-LID demonstrates a dynamic range of detection of at least 3 decades and coefficients of precision better than 20%. Finally, the spectral library was used to search for endogenous kinases in human cellular extract.

International audience; For the first time, quantitative electrospray (ESI) ionization efficiencies (IE), expressed as logIE values, obtained on different mass-spectrometric setups (four mass analyzers and four ESI sources) are compared for 15 compounds of diverse properties. The general trends of change of IE with molecular structure are the same with all experimental setups. The obtained IE scales could be applied on different setups: there were no statistically significant changes in the order of ionization efficiency and the root mean of squared differences of the logIE values of compounds between the scales compiled on different instruments were found to be between 0.21 and 0.55 log units. The results show that orthogonal ESI source geometry gives better differentiating power and additional pneumatic assistance improves it even more. It is also shown that the ionization efficiency values are transferable between different mass-spectrometric setups by three anchoring points and a linear model. The root mean square error of logIE prediction ranged from 0.24 to 0.72 depending on the instrument. This work demonstrates for the first time the inter-instrument transferability of quantitative electrospray ionization efficiency data.

International audience; Mass spectrometry-based methods have made significant progress in characterizing post-translational modifications in peptides and proteins; however, certain aspects regarding fragmentation methods must still be improved. A good technique is expected to provide excellent sequence information, locate PTM sites, and retain the labile PTM groups. To address these issues, we investigate 10.6 μm IRMPD, 213 nm UVPD, and combined UV and IR photodissociation, known as HiLoPD (high-low photodissociation), for phospho-, sulfo-, and glyco-peptide cations. IRMPD shows excellent backbone fragmentation and produces equal numbers of N- and C-terminal ions. The results reveal that 213 nm UVPD and HiLoPD methods can provide diverse backbone fragmentation producing a/x, b/y, and c/z ions with excellent sequence coverage, locate PTM sites, and offer reasonable retention efficiency for phospho- and glyco-peptides. Excellent sequence coverage is achieved for sulfo-peptides and the position of the SO3 group can be pinpointed; however, widespread SO3 losses are detected irrespective of the methods used herein. Based on the overall performance achieved, we believe that 213 nm UVPD and HiLoPD can serve as alternative options to collision activation and electron transfer dissociations for phospho- and glyco-proteomics.

http://www.societechimiquedefrance.fr/IMG/pptx/programme_journee_thematique.pptx; National audience

Ionization efficiency and mechanism in ESI is strongly affected by the properties of mobile phase. The use of mobile-phase properties to accurately describe droplets in ESI source is convenient but may be inadequate as the composition of the droplets is changing in the plume due to electrochemical reactions occurring in the needle tip as well as continuous drying and fission of droplets. Presently, there is paucity of research on the effect of the polarity of the ESI mode on mobile phase composition in the droplets. In this paper, the change in the organic solvent content, pH, and droplet size are studied in the ESI plume in both ESI+ and ESI– ionization mode. We introduce a rigorous way – the absolute pH (pHabs H 2 O) – to describe pH change in the plume that takes into account organic solvent content in the mobile phase. pHabs H 2 O enables comparing acidities of ESI droplets with different organic solvent contents. The results are surprisingly similar for both ionization modes, indicating that the dynamics of the change of mobile-phase properties is independent from the ESI mode used. This allows us to conclude that the evolution of ESI droplets first of all proceeds via the evaporation of the organic modifier and to a lesser extent via fission of smaller droplets from parent droplets. Secondly, our study shows that qualitative findings related to the ESI process obtained on the ESI+ mode can almost directly be applied also in the ESI– mode.

International audience; For the first time, the electrospray ionization efficiency (IE) scales in positive and negative mode are united into a single system enabling,direct comparison of IE values across ionization modes. This is made possible by the use of a reference Compound that ionizes to a similar extent in both positive, and negative modes. Thus, choosing the optimal (i.e., most sensitive) ionization conditions for a given set of analytes is enabled. Ionizatiori efficiencies of 33 compounds ionizing in both modes demonstrate that, contrary to general practice, negative mode allows, better sensitivity for 46% of such compounds Whereas the positive mode is preferred for only 18%, and for 36%, the results for both modes are comparable.

International audience; We describe the use of an extrinsic fluorophore (Rhodamine B isothiocyanate) as a versatile probe to measure rotational motions of proteins. To illustrate the usefulness of this probe, we describe the fluorescence anisotropy values of this fluorophore covalently linked to myoglobin protein measured in aqueous solutions of increased methanol content. Methanol-induced unfolding is revealed by the transition from constrained to free rotation of the covalently attached rhodamine B fluorophore. Graphical abstract Constrained to Free a) b) MGLSDGEWQQVLNVWGKVEA DIAGHGQEVLIRLFTGHPET LEKFDKFKHLKTEAEMKASE DLKKHGTVVLTALGGILKKK GHHEAELKPLAQSHATKHKI PIKYLEFISDAIIHVLHSKH PGDFGADAQGAMTKALELFR NDIAAKYKELGFQG 2 Introduction

International audience; UV photodissociation of proline-containing peptide ions leads to unusual product ions. In this paper, we report laser-induced dissociation of a series of proline-containing peptides at 213 nm. We observe specific fragmentation pathways corresponding to the formation of (y-2), (a + 2) and (b + 2) fragment ions. This was not observed at 266 nm or for peptides which do not contain proline residues. In order to obtain insights into the fragmentation dynamics at 213 nm, a small peptide (RPK for arginine-proline-lysine) was studied both theoretically and experimentally. Calculations of absorption spectra and non-adiabatic molecular dynamics (MD) were made. Second and third excited singlet states, S2 and S3, lie close to 213 nm. Non-adiabatic MD simulation starting from S2 and S3 shows that these transitions are followed by C-C and C-N bond activation close to the proline residue. After this first relaxation step, consecutive rearrangements and proton transfers are required to produce unusual (y-2), (a + 2) and (b + 2) fragment ions. These fragmentation mechanisms were confirmed by H/D exchange experiments.

RATIONALE To demonstrate the potential use of zebra fish (Danio rerio) as a model vertebrate organism by producing two-dimensional ion images of the whole zebra fish, and being able to distinguish particular areas of interest such as the brain, spinal cord, and stomach region using a desorption electrospray ionization (DESI) ion source coupled to a linear ion trap. METHODS Imaging experiments are performed on 45 µm sagittal slices of zebra fish (Danio rerio), which are thaw-mounted onto microscope glass slides. The slides are then analyzed using a solvent of acetonitrile/dimethylformamide (50:50) (ACN/DMF), with a solvent flow rate of 1.5 μL/min; data are acquired in negative ion mode. Raw mass spectrum data files are converted into a readable file for Biomap. The images produced are then analyzed for ion distributions. RESULTS We are able to create clear, distinct, chemical intensity images of the brain, spinal cord, and stomach based on lipid content as well as bile salt. The identities of these compounds were confirmed by tandem mass spectrometric (MS/MS) experiments and comparisons with literature. CONCLUSIONS Imaging of whole zebra fish is possible using ambient ionization techniques such as DESI. Analyses are fast and reliable. For most of the compounds observed, the identification by MS/MS can be performed directly from the fish tissue sample. Copyright © 2014 John Wiley & Sons, Ltd.

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International audience; Rationale: Tandem mass spectrometry (MS/MS) is the pivotal tool for protein structural characterization and quantification. Identification relies on the fragmentation step of tryptic peptides in bottom‐up strategy. Specificity of fragmentation can be obtained using laser‐induced dissociation (LID) in the visible range, after tagging of the targeted peptides with an adequate chromophore. Backbone fragmentation is required to obtain specific fragments and confident identification. We present herein a study of fragmentation patterns of chromophore‐tagged peptides in LID, showing the potential of LID methodology to provide the maximum number of fragments for further identification and quantification.Methods: A total of 401 cysteine‐containing tryptic peptides originating from the human proteome were derivatizated on the thiol group of cysteine with a Dabcyl maleimide chromophore, which has a high photo‐absorption cross section at 473 nm. The derivatized peptides were then analyzed by LID at 473 nm on a Q Exactive instrument.Results: LID spectra present a characteristic fragment at m/z 252.112 for all precursors. This product ion arises from the internal dissociation of the Dabcyl chromophore. Several peptide‐backbone fragment ions are also detected. Results show the quasi absence of fragmentation at the cysteine site. This indicates that part of the energy must be redistributed across the entire system despite excitation initially localized at the chromophore. Indeed, the fragmentation mainly occurs at 3 to 5 amino acids from the derivatized cysteine residue.Conclusions: LID of derivatized cysteine‐containing peptides displays the initial fragmentation of the chromophore. As energy is redistributed all along the peptide sequence, fragmentation of the peptide backbone is also observed. Thus, LID of chromophore‐tagged peptides produces adequate fragment ions, allowing both good sequence coverage for a greater confidence of identification, and a large choice of transitions for specific quantification.

International audience; Tandem mass spectrometry of co-oligomers composed of methacrylic acid (MAA) and methyl methacrylate (MMA) units was performed after activated electron photo-detachment dissociation (activated EPD). In this technique, doubly charged MAA/MMA co-oligomers were first produced in negative mode electrospray ionization. Species produced upon electron photo-detachment (220 nm) of these doubly charged co-oligomers were observed to spontaneously eliminate a carbon dioxide molecule. The so-formed radical anions were then activated by collision. In contrast to CID of negatively charged MAA/MMA co-oligomers, which does not provide informative data with regards to the end-groups, activated EPD is shown here to promote efficient radical-induced dissociation reactions thanks to the oxidation of a carboxylate pendant group upon laser irradiation. Product ions generated after backbone bond cleavages only contained the anionic initiating group. Combining these MS/MS results with the sum of the end-group masses determined from MS data allowed the terminating group to be characterized. As compared to activated EPD data obtained for a PMAA homopolymer holding the same end-groups, an additional product ion series was generated in the case of MAA/MMA copolymers, strongly suggesting that the hydrogen bond network established between neighboring MAA monomers is disrupted when M MA units are inserted in the polymeric backbone.

International audience; Single-photon, two-electron photodetachment from nickel phthalocyanine tetrasulfonic acid tetra anions, [NiPc](4-), was examined in the gas-phase using a linear ion trap coupled to the DESIRS VUV beamline of the SOLEIL Synchrotron. This system was chosen since it has a low detachment energy, known charge localization, and well-defined geometrical and electronic structures. A threshold for two-electron loss is observed at 10.2 eV, around 1 eV lower than previously observed double detachment thresholds on multiple charged protein anions. The photodetachment energy of [NiPc](4-) has been previously determined to be 3.5 eV and the photodetachment energy of [NiPc](3-center dot) is determined in this work to be 4.3 eV. The observed single photon double electron detachment threshold is hence 5.9 eV higher than the energy required for sequential single electron loss. Possible mechanisms are for double photodetachment are discussed. These observations pave the way toward new, exciting experiments for probing double photodetachment at relatively low energies, including correlation measurements on emitted photoelectrons.

Metalloenzymes preorganize the reaction environment to steer substrate(s) along the required reaction coordinate. Here, we show that phosphine ligands selectively facilitate protonation of binuclear silver hydride cations, [LAg2(H)]+ by optimizing the geometry of the active site. This is a key step in the selective, catalysed extrusion of carbon dioxide from formic acid, HO2CH, with important applications (for example, hydrogen storage). Gas-phase ion-molecule reactions, collision-induced dissociation (CID), infrared and ultraviolet action spectroscopy and computational chemistry link structure to reactivity and mechanism. [Ag2(H)]+ and [Ph3PAg2(H)]+ react with formic acid yielding Lewis adducts, while [(Ph3P)2Ag2(H)]+ is unreactive. Using bis(diphenylphosphino)methane (dppm) reshapes the geometry of the binuclear Ag2(H)+ scaffold, triggering reactivity towards formic acid, to produce [dppmAg2(O2CH)]+ and H2. Decarboxylation of [dppmAg2(O2CH)]+ via CID regenerates [dppmAg2(H)]+. These gas-phase insights inspired variable temperature NMR studies that show CO2 and H2 production at 70 °C from solutions containing dppm, AgBF4, NaO2CH and HO2CH., Designing catalysts and understanding the influence of ligands for particular transformations remains a highly challenging task. Here, the authors show that bisphosphine ligands can alter the geometry of the active site in silver catalysts, driving protonation and ultimately extrusion of carbon dioxide from formic acid.

Herein we report the successful implementation of the consecutive and simultaneous photodissociation with high (213 nm) and low (10.6 μm) energy photons (HiLoPD, high-low photodissociation) on ubiquitin in a quadrupole-Orbitrap mass spectrometer. Absorption of high-energy UV photon is dispersed over the whole protein and stimulates extensive C–Cα backbone fragmentation, whereas low-energy IR photon gradually increases the internal energy and thus preferentially dissociates the most labile amide (C–N) bonds. We noticed that simultaneous irradiation of UV and IR lasers on intact ubiquitin in a single MS/MS experiment provides a rich and well-balanced fragmentation array of a/x, b/y, and z ions. Moreover, secondary fragmentation from a/x and z ions leads to the formation of satellite side-chain ions (d, v, and w) and can help to distinguish isomeric residues in a protein. Implementation of high-low photodissociation in a high-resolution mass spectrometer may offer considerable benefits to promote a comprehensive portrait of protein characterization. Graphical Abstract ᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-016-1419-8) contains supplementary material, which is available to authorized users.

We thank Vincent Joly and Xavier Dagany for the development of data acquisition for fluorescence and Laure Cohendet and Mathilde Ghafar (MSc student) for their contribution to this work. Jacques Maurelli and Christian Clavier are acknowledged for their invaluable technical assistance.; International audience; Nanoparticle-based temperature imaging is an emerging field of advanced applications. Herein, the sensitivity of the fluorescence of rhodamine B-doped latex nanoparticles toward temperature is described. Submicrometer size latex particles were prepared by a surfactant-free emulsion polymerization method that allowed a simple and inexpensive way to incorporate rhodamine B into the nanoparticles. Also, rhodamine B-coated latex nanoparticles dispersed in water were prepared in order to address the effect of the dye location in the nanoparticles on their temperature dependence. A better linearity of the temperature dependence emission of the rhodamine B-embedded latex particles, as compared to that of free rhodamine B dyes or rhodamine B-coated latex particles, is observed. Temperature-dependent fluorescence measurements by fluorescent confocal microscopy on individual rhodamine B-embedded latex particles were found similar to those obtained for fluorescent latex nanoparticles in solution, indicating that these nanoparticles could be good candidates to probe thermal processes as nanothermometers.

International audience; The present paper describes the successful one-pot synthesis of self-stabilized particles composed of amphiphilic block copolymers based on poly(methacrylic acid) (PMAA) obtained by polymerization-induced self-assembly. First, controlled radical polymerization of MAA is performed in water using the RAFT process by taking advantage of our recent results showing the successful RAFT polymerization of MAA in water [Chaduc Macromolecules 2012, 45, 1241−1247]. The so-formed hydrophilic macroRAFT agents are then chain-extended in situ with a hydrophobic monomer to form amphiphilic block copolymer chains of controlled molar mass that self-assemble into stable nanoparticles. Various parameters such as the pH, the molar mass and the concentration of the PMAA segments or the nature of the hydrophobic block have been investigated.

International audience; Manipulation for simplifying or increasing the observed charge state distributions of proteins can be highly desirable in mass spectrometry experiments. In the present work, we implemented a vapor introduction technique to an Agilent Jet Stream ESI (Agilent Technologies, Santa Clara, CA, USA) source. An apparatus was designed to allow for the enrichment of the nitrogen sheath gas with basic vapors. An optical setup, using laser-induced fluorescence and a pH-chromic dye, permits the pH profiling of the droplets as they evaporate in the electrospray plume. Mechanisms of pH droplet modification and its effect on the protein charging phenomenon are elucidated. An important finding is that the enrichment with basic vapors of the nitrogen sheath gas, which surrounds the nebulizer spray, leads to an increase in the spray current. This is attributed to an increase in the electrical conductivity of water-amine enriched solvent at the tip exit. Here, the increased current results in a generation of additional electrolytically produced OH– ions and a corresponding increase in the pH at the tip exit. Along the electrospray plume, the pH of the droplets increases due to both droplet evaporation and exposure to basic vapors from the seeded sheath gas. The pH evolution in the ESI plume obtained using pure and basic seeded sheath gas was correlated with the evolution of the charge state distribution observed in mass spectra of proteins, in the negative ion mode. Taking advantage of the Agilent Jet Stream source geometry, similar protein charge state distributions and ion intensities obtained with basic initial solutions, can be obtained using native solution conditions by seeding the heated sheath gas with basic vapors.

International audience; Characterization of end-groups in poly(methacrylic acid) (PMAA) was achieved using tandem mass spectrometry after activated-electron photodetachment dissociation (activated-EPD). In this technique, multiply deprotonated PMAA oligomers produced in the negative-ion mode of electrospray ionization were oxidized into radical anions upon electron photodetachment using a 220 nm laser wavelength, and further activated by collision. In contrast to conventional collision induced dissociation of negatively charged PMAA, which mainly consists of multiple dehydration steps, fragmentation of odd-electron species is shown to proceed via a radical-induced decarboxylation, followed by reactions involving backbone bond cleavages, giving rise to product ions containing one or the other oligomer termination. A single radical-induced mechanism accounts for the four main fragment series observed in MS/MS. The relative position of the radical and of the anionic center in distonic precursor ions determines the nature of the reaction products. Experiments performed using PMAA sodium salts allowed us to account for relative abundances of product ions in series obtained from PMAA, revealing that ion stability is ensured by hydrogen bonds within pairs of MAA units.

International audience; A laser-induced fluorescence set-up profiling physico-chemical parameters of droplets and their changes in an electrospray plume is coupled to an Agilent single quadrupole mass spectrometer equipped with an Agilent Jet Stream electrospray source. This coupling allows investigating the evolution of the pH of droplets as they evaporate in the electrospray plume by measuring emission spectra of a pH-chromic dye. Simultaneous measurement of mass spectra and optical pH profiling permits to study the relation between the charge states of peptide anions in mass spectra and pH changes in the spray plume. In particular, due to changes in droplets evaporation rates, the source parameters (i.e., sheath gas flow and temperature) influence the pH of the droplets and relative intensities of charge states in peptide mass spectra.

Tandem mass spectrometry of poly(styrene sulfonate sodium salt) (PSS) was performed after activated electron photo-detachment dissociation (activated EPD). In this technique, doubly charged PSS oligomers were first produced in negative mode electrospray ionization, then oxidized into radical anions upon electron photo-detachment using a 220 nm laser wavelength, and further activated by collision. In contrast to the collision-induced dissociation (CID) of negatively charged PSS oligomers, which does not provide informative data with regard to the end-groups, activated-EPD is shown here to promote radical-induced dissociation reactions thanks to the oxidation of a sulfonate group upon laser irradiation. Major product ions generated after backbone bond cleavages contained one or the other chain terminations and could be accounted for by two main mechanisms. Moreover, each of the proposed dissociation reactions was shown to generate two distinct fragments, depending on the location of the oxidized monomer near one or the other chain terminal moieties. As a result, a combination of these two fragments allowed a straightforward mass characterization of each end-group.

Desorption electrospray ionization (DESI) mass spectrometry (MS) is used in an imaging mode to interrogate the lipid profiles of 15 μm thin tissue cross sections of injured rat spinal cord and normal healthy tissue. Increased relative intensities of fatty acids, diacylglycerols, and lysolipids (between +120% and +240%) as well as a small decrease in intensities of lipids (-30%) were visualized in the lesion epicenter and adjacent areas after spinal cord injury. This indicates the hydrolysis of lipids during the demyelination process due to activation of phospholipase A(2) enzyme. In addition, signals corresponding to oxidative degradation products, such as prostaglandin and hydroxyeicosatetraenoic acid, exhibited increased signal intensity by a factor of 2 in the negative ion mode in lesions relative to the normal healthy tissue. Analysis of malondialdehyde, a product of lipid peroxidation and marker of oxidative stress, was accomplished in the ambient environment using reactive DESI mass spectrometry imaging. This was achieved by electrospraying reagent solution containing dinitrophenylhydrazine as high-velocity charged droplets onto the tissue section. The hydrazine reacts selectively and rapidly with the carbonyl groups of malondialdehyde, and signal intensity of twice the intensity was detected in the lesions compared to healthy spinal cord. With a small amount of tissue sample, DESI-MS imaging provides information on the composition and distribution of specific compounds (limited by the occurrence of isomeric lipids with very similar fragmentation patterns) in lesions after spinal cord injury in comparison with normal healthy tissue allowing identification of the extent of the lesion and its repair.

Imaging mass spectrometry allows for the direct investigation of tissue samples to identify specific biological compounds and determine their spatial distributions. Desorption electrospray ionization (DESI) mass spectrometry has been used for the imaging and analysis of rat spinal cord cross sections. Glycerophospholipids and sphingolipids, as well as fatty acids, were detected in both the negative and positive ion modes and identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation and accurate mass measurements. Differences in the relative abundances of lipids and free fatty acids were present between white and gray matter areas in both the negative and positive ion modes. DESI-MS images of the corresponding ions allow the determination of their spatial distributions within a cross section of the rat spinal cord, by scanning the DESI probe across the entire sample surface. Glycerophospholipids and sphingolipids were mostly detected in the white matter, while the free fatty acids were present in the gray matter. These results show parallels with reported distributions of lipids in studies of rat brain. This suggests that the spatial intensity distribution reflects relative concentration differences of the lipid and fatty acid compounds in the spinal cord tissue. The "butterfly" shape of the gray matter in the spinal cord cross section was resolved in the corresponding ion images, indicating that a lateral resolution of better than 200 mum was achieved. The selected ion images of lipids are directly correlated with anatomic features on the spinal cord corresponding to the white and the gray matter.

Characterization of block size in poly(ethylene oxide)-b-poly(styrene) (PEO-b-PS) block copolymers could be achieved by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after scission of the macromolecules into their constituent blocks. The performed hydrolytic cleavage was demonstrated to specifically occur on the targeted ester function in the junction group, yielding two homopolymers consisting of the constitutive initial blocks. This approach allows the use of well-established MALDI protocols for a complete copolymer characterization while circumventing difficulties inherent to amphiphilic macromolecule ionization. Although the labile end-group in PS homopolymer was modified by the MALDI process, PS block size could be determined from MS data since polymer chains were shown to remain intact during ionization. This methodology has been validated for a PEO-b-PS sample series, with two PEO of number average molecular weight (Mn) of 2000 and 5000 g mol−1 and Mn(PS) ranging from 4000 to 21,000 g mol−1. Weight average molecular weight (Mw), and thus polydispersity index, could also be reached for each segment and were consistent with values obtained by size exclusion chromatography. This approach is particularly valuable in the case of amphiphilic copolymers for which Mn values as determined by liquid state nuclear magnetic resonance might be affected by micelle formation. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3380–3390, 2009

Different cationic adducts of poly(ethylene oxide)/polystyrene block co-oligomers could be produced by adjusting the salt concentration in the mobile phase using a coupling between liquid chromatography at critical conditions and electrospray ionization mass spectrometry. Formation of doubly lithiated adducts was observed at high LiCl concentration (1 mM) while lowering the salt concentration down to 0.1 mM allowed co-oligomers to be ionized with both a proton and a lithium. The fragmentation pathways observed to occur upon collision-induced dissociation of ionized copolymers were shown to be highly dependent on the nature of the cationic adducts. As a result, complementary structural information could be reached by performing MS/MS experiments on different ionic forms of the same co-oligomer molecule. On one hand, release of the nitroxide end-group as a radical from [M+2Li](2+) was followed by a complete depolymerization of the polystyrene block, allowing both this end-group and the polystyrene segment size to be determined. On the other hand, [M+H+Li](2+) precursor ions mainly dissociated via reactions involving bond cleavages within the nitroxide moiety, yielding useful structural information on this end-group.

The role of cations in the fragmentation of polymer nitroxide labile end groups, as often deplored in matrix-assisted laser desorption/ionization, was investigated. A combination of different spectroscopic techniques and theoretical calculations was implemented to address the mechanistic aspect of the end-group cleavage in a nitroxide-terminated poly(ethylene oxide) macroinitiator agent upon activation in mass spectrometry. When occurring during the ionization process, the homolytic cleavage of a C−ON bond in the end group results in the release of a nitroxide moiety and prevents the intact oligomer from being observed. In contrast, electrospray allowed oligomer ionization in a more gentle process and was used here in conjunction with tandem mass spectrometry to identify factors influencing the release of the end group. Collision-induced dissociation of intact electrosprayed oligomers showed that the elimination of the nitroxide radical depends on the cationizing agent. This fragmentation readily occurs f...

Coupling of liquid chromatography at critical conditions (LCCC) with on-line mass spectrometry (MS) detection was implemented via an electrospray ionization (ESI) interface, using a mobile phase containing the cationizing agent. Critical conditions established for poly(ethylene oxide) were used to characterize a poly(ethylene oxide)/polystyrene block copolymer (PEO-b-PS) in both MS and MS/MS modes. As co-oligomer molecules were successfully separated according to the PS block size, structural information could be reached from simplified MS spectra. The microstructure of this copolymer, synthesized by nitroxide-mediated polymerization, could further be unambiguously characterized in LCCC/ESI-MS/MS experiments since the PS block size could be reached by both the co-oligomer chromatographic behavior and its MS/MS pattern.

International audience; Characterization of acidic peptides and proteins is greatly hindered due to lack of suitable analytical techniques. Here we present the implementation of 213 nm ultraviolet photodissociation (UVPD) in high-resolution quadrupole-Orbitrap mass spectrometer in negative polarity for peptide anions. Radical-driven backbone fragmentation provides 22 distinctive fragment ion types, achieving the complete sequence coverage for all reported peptides. Hydrogen-deficient radical anion not only promotes the cleavage of Cα–C bond but also stimulates the breaking of N–Cα and C–N bonds. Radical-directed loss of small molecules and specific side chain of amino acids are detected in these experiments. Radical containing side chain of amino acids (Tyr, Ser, Thr, and Asp) may possibly support the N–Cα backbone fragmentation. Proline comprising peptides exhibit the unusual fragment ions similar to reported earlier. Interestingly, basic amino acids such as Arg and Lys also stimulated the formation of abundant b and y ions of the related peptide anions. Loss of hydrogen atom from the charge-reduced radical anion and fragment ions are rationalized by time-dependent density functional theory (TDDFT) calculation, locating the potential energy surface (PES) of ππ* and repulsive πσ* excited states of a model amide system.

International audience; We investigated how the temperature and size of charged droplets are affected by the electrospray ionization (ESI) process, using in situ measurements involving laser-induced fluorescence and Mie scattering on a thermal gradient focusing ESI source. Rhodamine dyes were employed as temperature indicators using ratiometric intensity-based fluorescence techniques. The results were compared to lifetime-based techniques using tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate, [Ru(bpy)3]2+. Both methods gave similar profiles. Nevertheless, the precision and sensitivity were higher for lifetime-based techniques in comparison to intensity-based techniques. Global warming (with ΔT ∼10 K) of the ESI plume is reported while the size of the droplet decreases along the plume. The global warming indicates that the conductive thermal transfer (between the superheated sheath gas and the solvent) is predominant and stronger than the cooling effect due to the evaporation of the droplets, and this outcome is effectively reproduced by a diffusion-controlled evaporation model. Thermal gradient focusing ESI sources therefore appear to be efficient sources for evaporating large amounts of solvent, along with an increase in temperature.

The coupling of normal-phase liquid chromatography to tandem mass spectrometry, previously developed in our laboratory, has been applied to the analysis of amitrole. This coupling utilizes an electrospray interface modified to accommodate the introduction of a make-up solution at the tip of the electrospray probe. A methanolic solution containing 3 mM ammonium acetate delivered at a flow rate of 10 µL · min−1 was found to be the optimal sheath liquid to promote successful ionization of the amitrole. Protonated molecules, arising from in-source dissociation of ammonium adducts, were subjected to tandem mass spectrometric experiments in a triple-quadrupole instrument. The main fragmentation reactions were characterized and selected to acquire chromatographic data in the multiple reaction monitoring mode. The limit of detection for amitrole was in the ppm range without any preconcentration step. Enhanced efficiency of ion transmission achievable nowadays in mass spectrometers (this analytical configuration was developed with a 15-year-old instrument) is reasonably expected to further improve this detection level. Copyright © 2006 John Wiley & Sons, Ltd.

International audience; Multistage mass spectrometry and density functional theory (DFT) were used to characterise the small silver hydride nanocluster, [Ag3H2L](+) (where L=(Ph2P)(2)CH2) and its gas-phase unimolecular chemistry. Collision-induced dissociation (CID) yields [Ag2HL](+) as the major product while laser-induced dissociation (LID) proceeds via H-2 formation and subsequent release from [Ag3H2L](+), giving rise to [Ag3L](+) as the major product. Deuterium labelling studies on [Ag3D2L](+) prove that the source of H-2 is from the hydrides and not from the ligand. Comparison of TD-DFT absorption patterns obtained for the optimised structures with action spectroscopy results, allows assignment of the measured features to structures of precursors and products. Molecular dynamics "on the fly" reveal that AgH loss is favoured in the ground state, but H-2 formation and loss is preferred in the first excited state S-1, in agreement with CID and LID experimental findings. This indicates favourable photo-induced formation of H-2 and subsequent release from [Ag3H2L](+), an important finding in context of metal hydrides as a hydrogen storage medium, which can subsequently be released by heating or irradiation with light.

International audience; Electrospray (ESI) ionization efficiencies (IE) of a set of 10 compounds differing by chemical nature, extent of ionization in solution (basicity), and by hydrophobicity (tetrapropylammonium and tetraethylammonium ion, triethylamine, 1-naphthylamine, N,N-dimethylaniline, diphenylphthalate, dimethylphtahalate, piperidine, pyrrolidine, pyridine) have been measured in seven mobile phases (three acetonitrile percentages 20%, 50%, and 80%, and three different pH-adjusting additives, 0.1% formic acid, 1 mM ammonia, pH 5.0 buffer combination) using the relative measurement method. MS parameters were optimized separately for each ion. The resulting relative IE data were converted into comparable logIE values by anchoring them to the logIE of tetrapropylammonium ion taking into account the differences of ionization in different solvents and thereby making the logIE values of the compounds comparable across solvents. The following conclusions were made from analysis of the data. The compounds with pK(a) values in the range of the solution pH values displayed higher IE at lower pH. The sensitivity of IE towards pH depends on hydrophobicity being very strong with pyridine, weaker with N,N-dimethylaniline, and weakest with 1-naphthylamine. IEs of tetraalkylammonium ions and triethylamine were expectedly insensitive towards solution pH. Surprisingly high IEs of phthalate esters were observed. The differences in solutions with different acetonitrile content and similar pH were smaller compared with the pH effects. These results highlight the importance of hydrophobicity in electrospray and demonstrate that high hydrophobicity can sometimes successfully compensate for low basicity.

Oral estrogens are directly associated with changes in plasma levels of coagulation proteins. Thus, the detection of any variation in protein concentrations due to estrogen contraceptives, by a simultaneous analysis of both coagulation proteins and estrogens, would be a very informative tool. In the present study, the merit of photo-selected reaction monitoring (SRM), a new analytical tool, was evaluated towards estrogens detection in plasma. Then, SRM and photo-SRM detection modes were combined for the simultaneous analysis of estrogen molecules together with heparin co-factor and factor XIIa, two proteins involved in the coagulation cascade. This study shows that photo-SRM could open new multiplexed analytical routes. Keywords: Estrogen, Coagulation factor protein, Metabolite, Photo-dissociation fragmentation, SRM