Search

International audience; The reactions of the medicinal gold(i) compound auranofin and its close analogues with vasopressin and the diselenide analogue were comparatively investigated by LC-electrospray MS/MS. Evidence is gained of the possible cleavage of the S-S and Se-Se bridges induced by Au(i). Notably, we found that, in the absence of reducing agents, the sulfur and selenium atoms are metallated only at high temperature (70 °C) with the preferential binding of gold to selenium. The reaction with the S-S bridge can take place at physiological temperature (37 °C) under reducing conditions. The implications of these results are discussed in the general frame of the reactivity of biologically relevant soft Lewis acids with peptides and proteins.

International audience; A rapid Ultra performance Liquid chromatography coupled with Quadrupole/time of flight Mass Spectrometry (UpLc-Qtof-MS) method was designed to quickly acquire high-resolution mass spectra metabolomics fingerprints for rosé wines. An original statistical analysis involving ion ratios, discriminant analysis, and genetic algorithm (GA) was then applied to study the discrimination of rosé wines according to their origins. After noise reduction and ion peak alignments on the mass spectra, about 14 000 different signals were detected. The use of an in-house mass spectrometry database allowed us to assign 72 molecules. Then, a genetic algorithm was applied on two series of samples (learning and validation sets), each composed of 30 commercial wines from three different wine producing regions of France. Excellent results were obtained with only four diagnostic peaks and two ion ratios. This new approach could be applied to other aspects of wine production but also to other metabolomics studies. Wine is a widely consumed alcoholic beverage with a high commercial value. More specifically, the worldwide consumption of rosé wine has increased by 20% since 2002 1. Because of its high commercial value, it can become a subject of fraud, and authenticity control is required in order to maintain wine quality and to detect any adulteration 2. Thousands of molecules can be found in wines, including polyphenols 3. Recently, more than one hundred polyphenols have been quantified in various rosé wines 4. They are key components involved in color, taste and quality of wines. Their amount and composition depend on many different factors such as grape variety, geographic origin, winemaking, age. Several methods have already been developed for wine authentication purpose 5. They can be divided into two categories: metabolite profiling 6-8 or metabolomic fingerprinting 9,10. The first one is a targeted analysis focusing on a limited number of representative components while the second one is a non-targeted approach. Both methods were applied to red or white wines. In a previous work 11 , a very fast UPLC-QTOF-MS method was developed to characterize red wines from different grape varieties. One specific ion ratio was used to discriminate commercial red wines from three grape varieties. In this paper, we focused on the influence of the geographic origin of some rosé French wines. The chemical composition of grapes depends on the sum of different environmental conditions, which can be defined as a "terroir" that should influence the grape and wine composition. The goals of this paper were to develop: • A new and very fast UPLC-QTOF-MS wine metabolomics method with a focus on wine pigments. • An original statistical method and workflow that allow the robust discrimination of rosés wines according to their origins by using mass spectrometry ion ratio fingerprints. Results and Discussions UPLC-QTOF-MS analysis. First, a fast UPLC-QTOF-MS method was developed to rapidly acquire high-resolution mass spectra. In accordance with previous work and conclusions, we have used a short gradient instead of isocratic elution conditions or direct injections 11. It was shown that the last two methods gave limited results probably due to ionization suppression effect. In this work, we chose to work on the positive ionization

International audience; Venom peptides are promising drug leads, but their therapeutic use is often limited by stability and bioavailability issues. In this study, we designed cyclic analogues of α-conotoxin CIA, a potent muscle nicotinic acetylcholine receptor (nAChR) blocker with a significantly lower affinity at the neuronal α3β2 subtype. Remarkably, all analogues retained the low nanomolar activity of native CIA toward muscle-type nAChRs but showed greatly improved resistance to degradation in human serum and, surprisingly, displayed up to 52-fold higher potency for the α3β2 neuronal nAChR subtype (IC50 1.3 nM). Comparison of nuclear magnetic resonance-derived structures revealed some differences that might explain the gain of potency at α3β2 nAChRs. All peptides were highly paralytic when injected into adult zebrafish and bath-applied to zebrafish larvae, suggesting barrier-crossing capabilities and efficient uptake. Finally, these cyclic CIA analogues were shown to be unique pharmacological tools to investigate the contribution of the presynaptic α3β2 nAChR subtype to the train-of-four fade.

Wine oxidation and ageing involve many complex chemical pathways and reaction mechanisms. The purpose of this study is to set up new and reproducible accelerated red wine ageing tests and identify chemical oxidation or ageing molecular markers. Three accelerated and reproducible ageing tests were developed: a heat test (60 &deg, C), an enzymatic test (laccase test, a chemical test (hydrogen peroxide test). Depending on the test, oxygen consumption was significantly different. For a young wine (2018), the oxygen consumption rate moved from 2.40 ppm.h&minus, 1 for the heat test to 3.33 ppm.h&minus, 1 for the enzymatic test and 2.86 ppm.h&minus, 1 for the chemical test. Once applied to two other vintages (2010 and 2014) from the same winery, the tests revealed different comportments corresponding to wine natural evolution. High resolution UPLC-MS was performed on forced ageing samples and compared to naturally aged red wines. Specific oxidation or ageing ion markers were found with significant differences between tests, revealing the specificity of each test and different possible molecular pathways involved. The hydrogen peroxide test seems to be closer to natural oxidation with an important decrease in absorbance at 520 nm and similar molecular ion variations for [M+H]+ = 291, 331, 347, 493, 535, 581, 639 Da.

RATIONALE Both amide bond protonation triggering peptide fragmentations and the controversial b2 -ion structures have been subjects of intense research. The involvement of histidine (H), with its imidazole side chain that induces specific dissociation patterns involving inter-side-chain (ISC) interactions, in b2 -ion formation was investigated, focusing on the QHS model tripeptide. METHODS To identify the effect of histidine on fragmentations issued from ISC interactions, QHS was selected for a comprehensive analysis of the pathways leading to the three possible b2 -ion structures, using quantum chemical calculations performed at the DFT/B3LYP/6-311+G* level of theory. Electrospray ionization ion trap mass spectrometry allowed the recording of MS2 and MS3 tandem mass spectra, whereas the Quantum Chemical Mass Spectrometry for Materials Science (QCMS2 ) method was used to predict fragmentation patterns. RESULTS Whereas it is very difficult to differentiate among protonated oxazolone, diketopiperazine, or lactam b2 -ions using MS2 and MS3 mass spectra, the calculations indicated that the QH b2 -ion (detected at m/z 266) is probably a mixture of the lactam and oxazolone structures formed after amide nitrogen protonation, making the formation of diketopiperazine less likely as it requires an additional step for its formation. CONCLUSIONS In contrast to glycine-histidine-containing b2 -ions, known to be issued from the backbone-imidazole cyclization, we found that interactions between the side chains were not obvious to perceive, neither from a thermodynamics nor from a fragmentation perspective, emphasizing the importance of the whole sequence on the dissociation behavior usually demonstrated from simple glycine-containing tripeptides.

International audience; New designer drugs, as synthetic cannabinoids (SCs), continuously appear on the market and are booming due to their cannabis-like effect. New generation of smokable SCs, structurally dissimilar from Δ9-tetrahydrocannabinol (THC), have isomers with distinguishable pharmacokinetic parameters and therefore different in vivo effects. The isoforms are misidentified using conventional techniques such as gas or liquid chromatography coupled to mass spectrometry - or tandem mass - spectrometry. The aim of this study was to differentiate three positional isomers (JWH-007, JWH-019 and JWH-122) in single human hair samples, which store numerous substances revealing a way of life and consumption style. Matrix-assisted laser desorption/ionization (MALDI) combined with imaging is an innovative and powerful tool used since few years, especially in forensic research. Herein, we propose an innovative method to monitor the drugs of abuse consumption through direct mapping of the compounds with a high spatial distribution in human hair samples, by state-of-art imaging MALDI-MSn. Three positional SC isomers (JWH-007, JWH-019 and JWH-122) were analysed using high and low fragmentation energy and the resulting MS/MS and even MS3 spectra differentiated the SCs. The MALDI-MS/MS and MS3 imaging was performed on hair soaked in a mixture of the three SCs as well as on hair from self-reported SC user, proving the potential of the technique for a forensic use. Keeping in mind that spatial distribution of organics from whole hair remains a challenge, the described methodology is a very promising analytical tool to probe the consumption of complex drugs and obtain correlation with its origin.

Glycosylated flavanols (monoglycosides and diglycosides) in skin and seed extracts of Vitis vinifera grapes grown in Castilla-La Mancha (Spain) were investigated using ultra-high-performance liquid chromatography&mdash, tandem mass spectrometry (UHPLC-ESI-QQQ-MS/MS). Six grape varieties (Air&eacute, n, Tempranillo, the recently identified Albillo Dorado, Montonera del Casar, Moribel, and Tinto Fragoso) were studied over two consecutive years (2016 and 2017). A total of twenty monomeric flavanol monoglycosides, four diglycosylated monomers, and three dimeric flavanol monoglycosides were detected in all grape samples. The diversity observed in the composition of glycosylated flavanol in the grape berries suggests a strong influence of variety and grape tissue (skin or seed). Monomeric flavanol glycosides were more abundant in grape seed extracts, in contrast with monoglycosylated dimeric forms. In addition, the glycosylated flavanol content was related to berry color in grape skins, with higher concentrations measured in black grape varieties.

International audience; In the search for an alternative strategy to the radioactivity measurement conventionally performed to probe receptor–ligand interactions in pharmacological assays, we demonstrated that selenium labeling of the studied ligand combined with elemental mass spectrometry was as efficient and robust as the reference method but devoid of its environmental and health hazards. The proof-of-concept was illustrated on two GPCR receptors, vasopressin (V1A) and cholecystokinin B (CCK-B), involving peptides as endogenous ligands. We proposed several methodologies to produce selenium-labeled ligands according to peptide sequences along with binding affinity constraints. A selection of selenopeptides that kept high affinities toward the targeted receptor were engaged in saturation and competitive binding experiments with subsequent sensitive RP-LC-ICP-MS measurements. Experimental values of affinity constant (Ki) were perfectly correlated to literature data, illustrating the general great potency of replacing radioactive iodine by selenium for ligand labeling to further undergo unaffected pharmacology experiments efficiently monitored by elemental mass spectrometry.

Monomeric and dimeric flavanol glycosides were analyzed in Vitis vinifera grapes and seeds during ripening. An analytical method using ultra-high performance liquid chromatography coupled with a triple quadrupole mass spectrometry (UHPLC-ESI-QQQ-MS/MS) in multiple reaction monitoring (MRM) mode was employed. Three grape varieties (Merlot, Syrah and Tannat) were chosen and grape berries were sampled at different stages of development. Ten monoglycosylated and six diglycosylated flavanol monomers were detected. Twelve monoglycosylated and three diglycosylated flavanol dimers were also detected for all three grape varieties. All diglycosides were detected for the first time in Vitis vinifera grapes, though some of these compounds were only detected in skins or seeds. Furthermore, the evolution of all these compounds was studied, and a decrease in monomeric (epi) catechin monoglycosides was observed during ripening for Tannat, Merlot and Syrah grape skins. The dimers would appear to accumulate in skin tissues up to mid-summer (after veraison) and decrease when grape berries reached maturity.

International audience; RATIONALE: Many important biological processes rely on specific biomarkers (such as metabolites, drugs, proteins or peptides, carbohydrates, lipids, ...) that need to be monitored in various fluids (blood, plasma, urine, cell cultures, tissue homogenates, …). Although mass spectrometry (MS) hyphenated to liquid chromatography (LC) is widely accepted as a 'gold-standard' method for identifying such synthetic chemicals or biological products, their robust fast sensitive detection from complex matrices still constitutes a highly challenging matter.METHODS: In order to circumvent the constraints intrinsic to LC/MS technology in terms of prior sample treatment, analysis time and overall method development to optimize ionization efficiency affecting the detection threshold, we investigated laser desorption/ionization mass spectrometry (LDI-MS) by directly depositing the sample under study onto cheap inert nanostructures made of silicon to perform straightforward sensitive and rapid screening of targeted low mass biomarkers on a conventional MALDI platform.RESULTS: The investigated silicon nanostructures were found to act as very efficient ion-promoting surfaces exhibiting high performance for the detection of different classes of organic compounds, including glutathione, glucose, peptides and antibiotics. Achieving such broad detection was compulsory to develop a SALDI-MS-based pre-screening tool.CONCLUSIONS: The key contribution of the described analytical strategy consists of designing inert surfaces that are fast (minute preparation) and cheap to produce, easy to handle and able to detect small organic compounds in matrix-free LDI-MS prerequisite for biomarkers pre-screening from body fluids without the recourse of any separation step.

In the search for new peptide ligands containing selenium in their sequences, we investigated l-4-selenazolidine-carboxylic acid (selenazolidine, Sez) as a proline analog with the chalcogen atom in the γ-position of the ring. In contrast to proteinogenic selenocysteine (Sec) and selenomethionine (SeMet), the incorporation within a peptide sequence of such a non-natural amino acid has never been studied. There is thus a great interest in increasing the possibility of selenium insertion within peptides, especially for sequences that do not possess a sulfur containing amino acid (Cys or Met), by offering other selenated residues suitable for peptide synthesis protocols. Herein, we have evaluated selenazolidine in Boc/Bzl and Fmoc/tBu strategies through the synthesis of a model tripeptide, both in solution and on a solid support. Special attention was paid to the stability of the Sez residue in basic conditions. Thus, generic protocols have been optimized to synthesize Sez-containing peptides, through the use of an Fmoc-Xxx-Sez-OH dipeptide unit. As an example, a new analog of the vasopressin receptor-1A antagonist was prepared, in which Pro was replaced with Sez [3-(4-hydroxyphenyl)-propionyl-d-Tyr(Me)-Phe-Gln-Asn-Arg-Sez-Arg-NH2]. Both proline and such pseudo-proline containing peptides exhibited similar pharmacological properties and endopeptidase stabilities indicating that the presence of the selenium atom has minimal functional effects. Taking into account the straightforward handling of Sez as a dipeptide building block in a conventional Fmoc/tBu SPPS strategy, this result suggested a wide range of potential uses of the Sez amino acid in peptide chemistry, for instance as a viable proline surrogate as well as a selenium probe, complementary to Sec and SeMet, for NMR and mass spectrometry analytical purposes.

Monomeric and dimeric flavanol glycosides were quantified by UHPLC-MRM in Syrah (SYR) and Grenache (GRE) grapes and in their corresponding wines for the first time. Quantities were extremely variable depending on grape tissue (seeds or skins) and during fermentation. Overall, 22 monomeric and dimeric mono- and diglycosides were determined with concentrations ranging from 0.7 nanograms to 0.700 micrograms per gram of grape tissue, and 0 to 60 micrograms per liter for wines. The evolution of the glycosides’ composition during winemaking suggests that almost all these compounds originate in the grapes themselves and display different extraction kinetics during winemaking. One isomer of the monomeric (epi) flavanol monoglycosides seemed to be biosynthesized by yeasts during wine fermentation. The sharp decrease in concentration of some isomers at the late stages of fermentation or after pressing suggests that some grape glycosidase activities convert these compounds into non-glycosylated flavanols.

We report in this piece of work the synthesis of biosilica hybrid materials able to develop from hydrophobic polyesters to intermediate hydrophilic polyesters and then to polyamides through an O–N acyl transfer shift. After demonstration of the O–N acyl shift on a model depsipeptide anchored in the pores of ordered mesoporous silica (OMS), the preparation of three bio oligomer–silica hybrid materials is described. The oligomerisation of protected serine lactones was initiated in the pores of carboxylate functionalized OMS. Hybrid protected serine polyester–OMS were obtained and could be converted (i) into cationic polyesters, which could be switched (ii) into neutral polyamide–OMS in basic conditions through a multi O–N acyl shift.

International audience; In this paper, we report an original method to immobilize a model peptide on silicon nanowires (SiNWs) via a photolinker attached to the SiNWs’ surface. The silicon nanowires were fabricated by a metal assisted chemical etching (MACE) method. Then, direct characterization of the peptide immobilization on SiNWs was performed either by X-ray photoelectron spectroscopy (XPS) or by laser-desorption/ionization mass spectrometry (LDI-MS). XPS allowed us to follow the peptide immobilization and its photorelease by recording the variation of the signal intensities of the different elements present on the SiNW surface. Mass spectrometry was performed without the use of an organic matrix and peptide ions were produced via a photocleavage mechanism. Indeed, thanks to direct photorelease achieved upon laser irradiation, a recorded predictable peak related to the molecular peptide ion has been detected, allowing the identification of the model peptide. Additional MS/MS experiments confirmed the photodissociation site and confirmed the N-terminal immobilization of the peptide on SiNWs.

International audience; Radiolabeling of ligands is still the gold standard in the study of high-affinity receptor-ligand interactions. In an effort toward safer and simpler alternatives to the use of radioisotopes, we developed a quantitative and highly sensitive matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) method that relies on the use of chemically tagged ligands designed to be specifically detectable when present as traces in complex biological mixtures such as cellular lysates. This innovative technology allows easy, sensitive detection and accurate quantification of analytes at the sub-nanomolar level. After statistical validation, we were able to perform pharmacological evaluations of G protein-coupled receptor (V1A-R)-ligand interactions. Both saturation and competitive binding assays were successfully processed.

International audience; We have evaluated the laser desorption ionization mass spectrometry (LDI-MS) performance of six nanostructured silicon surfaces of different morphologies and chemical functionalizations. The substrates have been synthesized either by metal-assisted etching method or by vapor−liquid−solid (VLS) growth technique. In addition to the commercial nanostructured silicon-based surface (NALDI) target plates, serving as reference, the homemade surfaces have been evaluated in mass spectrometry experiments conducted with peptide solutions mimicking tryptic digests. LDI surfaces synthesized by metal-assisted etching method were the most efficient in terms of signal intensities and number of detected peptides. The surface providing the best LDI-MS performance was composed of two nanostructured layers. Interestingly, we also observed a significant influence of the type of organic coating (hydrocarbon vs fluorocarbon) on peptide ionization discrimination.

International audience; We report on the simple application of a new nanostructured silicon (NanoSi) substrate as laser desorption/ionization (LDI)-promoting surface for high-throughput identification of protein tryptic digests by a rapid MS profiling and subsequent MS/MS analysis. The NanoSi substrate is easily prepared by chemical etching of crystalline silicon in NH4F/HNO3/AgNO3 aqueous solution. To assess the LDI performances in terms of sensitivity, repeatability and robustness, the detection of small synthetic peptides (380-1700 Da) was investigated. Moreover, peptide sequencing was tackled. Various tryptic synthetic peptide mixtures were first characterized in MS and MS/MS experiments carried out on a single deposit. Having illustrated the capability to achieve peptide detection and sequencing on these ionizing surfaces in the same run, protein tryptic digests from Cytochrome C, É¿-Casein, BSA and Fibrinogen were then analyzed in the femtomolar range (from 50 fmol for Cytochrome C down to 2 fmol for Fibrinogen). Comparison of the NanoSi MS and MS/MS data with those obtained with sample conditioned in organic matrix demonstrated a great behavior for low mass responses. We demonstrated the capability of LDI on NanoSi to be a complementary method to MALDI peptide mass fingerprinting ensuring determination of peptide molecular weights and sequences for more efficient protein database searches.

In the search for alternative inert surfaces replacing silicon chips in Desorption/Ionization On porous Silicon (DIOS)-like mass spectrometry analyses, nanostructured silicon-based NALDI™ chips were evaluated in Laser Desorption/Ionization (LDI) of peptides. Comparisons were made using commercially available DIOS chips (MassPREP™-DIOS-target™), amorphous carbon powder from lead pencil and porous silica gel used for chromatographic purposes as reference supports. A set of synthetic model peptides presenting variable amino acid sequences of various lengths was analyzed under all conditions. The LDI responses of the four ‘matrix-free’ techniques were compared, especially in terms of peptide detection sensitivity and overall experiment robustness. Copyright © 2009 John Wiley & Sons, Ltd.

Resol type resins were prepared in alkaline conditions (potassium hydroxide or potassium carbonate) using furfural obtained by acid hydrolysis of abundant renewable resources from agricultural and forestry waste residues. The structures of the resins were fully determined by 1H, 13C, and 2D NMR spectrometries with the help of four models compounds synthesized specially for this study. MALDI-Tof mass spectrometry experiments indicated that a majority of linear oligomers and a minority of cyclic ones constituted them. Composites were prepared with furfural–phenol resins and sisal fibers. These fibers were chosen mainly because they came from natural lignocellulosic material and they presented excellent mechanical properties. Thermal analyses (dTG and DSC) and electron microscopy images indicated that the composites displayed excellent adhesion between resin and fibers. Impact strength measurement showed that mild conditions were more suitable to prepare thermosets. Nevertheless, mild conditions induced a high-diffusion coefficient for water absorption by composites. Composites with good properties could be prepared using high proportion of materials obtained from biomass without formaldehyde. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Macromolecules of tunable solubility, used to mimic inert insoluble materials while maintaining solution conditions, allowed the performance of efficient supported organic chemistry and facilitated in situ reaction monitoring. To satisfy the high throughput requirements of automated synthetic processes, organic syntheses carried out on bifunctional polyethylene glycol polymers (PEG(3400)-OH) were monitored step-by-step by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A protocol was designed to control the ionization mechanism of such polymers exhibiting high affinity for alkali metal cations. Automated, rapid, and reliable data interpretation was performed by an in-house developed visual basic application relying on the sodiated ion accurate monoisotopic mass measurement. The methodology was illustrated through the monitoring of a six-step synthetic scheme.

International audience; We report applications of new hybrid organic-inorganic silica based materials as laser desorption/ionization (LDI)-promoting surfaces for high-throughput identification of peptides. The driving force of our work was to design a new material composed of a conventional MALDI matrix covalently attached to silica with a high organic/inorganic ratio in order to improve the UV absorption by such LDI hybrid matrices. Amorphous CHCA-functionalized silica presenting an organic content up to 1.3 mmol g 1 (around 40% in weight from TGA and elementary analysis measurements) gave very interesting LDI performances in terms of detection sensitivity as well as relative ionization discrepancy (spectral suppression) through the analyses of small synthetic peptide mixtures (550-1300 Da) taking CHCA and amorphous silica as model matrices for control experiments.

International audience; Mass spectrometry is a powerful analytical tool allowing rapid and sensitive structural elucidation of a wide range of molecules issued from solution-, solid- and liquid-phase syntheses. Therefore, mass spectrometry has become the most widely used tool to probe combinatorial libraries. A significant portion of the reported combinatorial data are being produced using solid phase organic synthesis. In contrast to indirect strategies where the tethered structures were released from the support into solution to undergo standard mass spectrometric analyses, static - secondary ion mass spectrometry (S-SIMS) has enabled the identification of support-bound molecules without any chemical treatment of the resin bead. Such non-destructive characterization was applied at the bead level and facilitated the step-by-step monitoring of solid-phase peptide syntheses. Side-reactions were also detected. The relevance of S-SIMS in the rehearsal phase of combinatorial chemistry is demonstrated by comparison with infrared and nuclear magnetic resonance (NMR) spectroscopies, the two other techniques investigated in that field. An alternative to solid-phase synthesis consists of assembling molecules on a soluble polymer. This methodology is termed liquid-phase synthesis. Compound characterization is facilitated since the derivatized support is soluble in spectroscopic solvents used in NMR or in electrospray ionization mass spectrometry. The advantages and drawbacks of this approach will be discussed in terms of the direct monitoring of supported reactions during chemistry optimization and rehearsal library validation.

A straightforward method for the preparation of hybrid bioorganic–inorganic materials is reported. Common strategies to synthesize such promising materials require special surface modifications of silica followed by grafting of the organic moiety via chemoselective ligation. In this context, we set up a general and bottom-up strategy relying on modified peptides functionalized with a trialkoxysilane group. Used in mixtures with TEOS and a surfactant as the structure directing agent, these hybrid building blocks allow one step direct synthesis of bioorganic–inorganic hybrid materials. Two examples were chosen to demonstrate our general approach. (1) An antifouling surface was prepared by dip coating of a sol containing an antibacterial silylated peptide. (2) Organized mesoporous silica displaying a peptide catalyst in the pores was prepared in one step and tested.

International audience; A straightforward synthesis of comb-polypeptides of repeated peptide sequences was developed. These polypeptides were obtained by ROP of defined NCA without any post-polymerization grafting. The key to this strategy relies on the preparation of pure NCA bearing a peptide sequence on its side chain, by an original solid supported methodology.

cited By 8; International audience; Despite correct purity of crude peptides prepared on trityl resin by Fmoc/tBu microwave assisted solid phase peptide synthesis, surprisingly, lower yields than those expected were obtained while preparing C-terminal acid peptides. This could be explained by cyclization/cleavage through diketopiperazine formation during the second amino acid deprotection and third amino acid coupling. However, we provide here evidence that this is not the case and that this yield loss was due to high temperature promoted hydrolysis of the 2-chlorotrityl ester, yielding premature cleavage of the C-terminal acid peptides. © 2013 Springer-Verlag Wien.

International audience; The straightforward synthesis of a double-lasso macrocycle from a nonsymmetrical [c2]daisy chain, using the copper(I)-catalyzed Huisgen alkyne azide 1,3-dipolar cycloaddition, is described. The preparation of the nonsymmetrical alkyne azide [c2]daisy chain precursor was realized in situ via the exchange of the monomers contained in both symmetrical alkyne and azide [c2]daisy chains and was followed by mass spectrometry.

International audience; In the search of new robust and environmental-friendly analytical methods able to answer quantitative issues in pharmacology, we explore liquid chromatography (LC) associated with elemental mass spectrometry (ICP-MS) to monitor peptides in such complex biological matrices. The novelty is to use mass spectrometry to replace radiolabelling and radioactivity measurements, which represent up-to now the gold standard to measure organic compound concentrations in life science. As a proof of concept, we choose the vasopressin (AVP)/V1A receptor system for model pharmacological assays. The capacity of ICP-MS to provide highly sensitive quantitation of metallic and hetero elements, whatever the sample medium, prompted us to investigate this technique in combination with appropriate labelling of the peptide of interest. Selenium, that is scarcely present in biological media, was selected as a good compromise between ICP-MS response, covalent tagging ability using conventional sulfur chemistry and peptide detection specificity. Applying selenium monitoring by elemental mass spectrometry in pharmacology is challenging due to the very high salt content and organic material complexity of the samples that produces polyatomic aggregates and thus potentially mass interferences with selenium detection. Hyphenation with a chromatographic separation was found compulsory. Noteworthy, we aimed to develop a straightforward quantitative protocol that can be performed in any laboratory equipped with a standard macrobore LC-ICP-MS system, in order to avoid time-consuming sample treatment or special implementation of instrumental set-up, while allowing efficient suppression of all mass interferences to reach the targeted sensitivity. Significantly, a quantification limit of 57 ng Se L-1 (72 femtomoles of injected Se) was achieved, the samples issued from the pharmacological assays being directly introduced into the LC-ICP-MS system. The established method was successfully validated and applied to the measurement of the vasopressin ligand affinity for its V1A receptor through the determination of the dissociation constant (Kd) which was compared to the one recorded with conventional radioactivity assays.

International audience; By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (bn-1+H2O) ion, corresponding to a shortened nonscrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H2OandNH3) and multiple backbone amide ruptures (b-type internal ions), the described Cterminal residue exclusion is highly identifiable giving raise to a single fragment ion in the high mass range of the MS/MS spectra. The mass difference between this signal and the protonated molecular ion corresponds to the mass of the C-terminal residue. It allowed a straightforward identification of the amino acid positioned at this extremity. It must be emphasized that a neutral residue loss can be misattributed to the formation of a ym-1 ion, i.e., to the loss of the N-terminal residue following the a1ym-1 fragmentation channel. Extreme caution must be adopted when reading the direct sequence ion on the positive ion MS/MS spectra of singly charged peptides not to mix up the attribution of the N- and C-terminal amino acids. Although such peculiar fragmentation behavior is of obvious interest for de novo peptide sequencing, it can also be exploited in proteomics, especially for studies involving digestion protocols carried out with proteolytic enzymes other than trypsin (Lys-N, Glu-C, and Asp-N) that produce arginine-containing peptides.

International audience; The present study was conducted to assess the structures of the main unknown oxygenated metabolites of EAPB0203. The first step was to assign all the 1H and 13C NMR of both EAPB0203 and its demethylated metabolite (EAPB0202) to the corresponding atoms in their molecular structures and to elucidate the fragmentation pathways for the [M + H] + ions of these compounds using high-resolution mass spectrometry (MS). MS/MS spectra showed that both protonated molecules possessing an even number of electrons were unexpectedly losing radicals such as H*,CH3*, or even C7H7* giving stable radical cations. In vitro metabolism studies were investigated in rat and dog liver microsomes and in the filamentous fungus Cunninghamella elegans. Structural elucidation of six oxygenated metabolites was performed based on the following: (i) their fragmentation pathways in liquid chromatography−MS/MS (LC-MS/MS) analyses; (ii) comparison of their changes in their molecular masses and fragment ions with those of the parent drugs; and (iii) the results of online H/D exchange experiments that provided additional evidence in differentiating hydoxylated metabolites from N-oxides. Structures of the metabolites were elucidated by LC-MS/MS and comparison with synthetic standards; structures of these standards were confirmed using one- and two-dimensional 1H NMR spectroscopies.

International audience; Sulfation of tyrosine residues is a key posttranslationalmodification in the regulation of various cellular processes. As such, the detection and localization of tyrosine sulfation is an essential step toward the elucidation of the physiological and pathological roles of this process. Despite substantial advances, intact sulfated peptides are still difficult to detect by MALDI-MS due to the extreme lability of the sulfo-moiety. The present report demonstrates for the first time how intact sulfated peptides can be directly and specifically detected by MALDI-MS in positive reflectronmode by using pyrenemethylguanidine (pmg) as a noncovalent derivatizing agent and an ionization enhancer. This new method allows the determination of the degree of sulfation of sulfopeptides pure or in mixtures. Moreover, the observation of specific peaks in the mass spectra enables a rapid and unambiguous discrimination between phospho- and sulfopeptides.

We report on the simple deposition of Stober silica nanoparticles (SiO 2 NPs) on conventional MALDI target plate for high throughput laser desorption/ionization mass spectrometry (LDI-MS) analyses of peptide mixtures with sensitivity in the femtomolar range. This low-cost easily prepared material allowed straightforward LDI experiments by deposition of the studied samples directly onto a pre-spotted MALDI plate. This analytical strategy can be performed in any laboratory equipped with a MALDI-TOF instrument. All key benefits of organic matrix-free technologies were satisfied while maintaining a high level of detection performances (sensitivity and reproducibility/repeatability). In particular, sample preparation was simple and detection in the low mass range was not hampered by matrix ions. Imaging studies were undertaken to query sample dispersion into the inert SiO 2 NPs and to help into the search of the best experimental conditions producing homogeneous analyte distribution within the deposit. In contrast to commercial disposable LDI targets designed for single use and requiring an adaptor such as NALDI™, the proposed SiO 2 NPs pre-spotting on a MALDI target plate allowed very easily switching between MALDI and LDI experiments. They can be conducted either simultaneously (positions with an organic matrix or SiO 2 NPs) or in the row (support prepared in advance, stored and washed after use). The overall cost and versatility of the methodology made it very attractive to MALDI users in many domains (peptidomics, proteomics, metabolomics).

cited By 3; International audience; The synthesis and use of novel polymer-supported reagents for disulfide bond formation is described. This family of supported reagents consists of a series of oxidized methionines grafted onto a solid support. Their cost and the simplicity of their preparation through N-carboxyanhydride polymerization on beads make them reactants of choice for the formation of disulfide bridges in peptides. Copyright © 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

International audience; In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola Frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion since a specific cleavage at the amide X-Lys chain is obtained providing N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a In lysine or arginine residues solely at the C-terminal position, and thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with ESI-QqTof and MALDI-Tof/Tof mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

International audience; In this paper, mesoporous organosilicas functionalised with aminopropyl groups have been successfully used for peptide oligomerisation. For this purpose, three mesoporous silica SBA-15 type containing different amounts of aminopropyl groups were prepared by direct synthesis and using 3- tert-butyloxycarbonylamino propyltriethoxysilane. Thanks to amino groups and under well selected experimental conditions, amino acids N-carboxyanhydride-polymerisation has been achieved within the pores with control of the physical properties of peptide functionalised hybrid materials. NCA of alanine, side chain protected glutamic acid and methionine were used for this study. For the first time, direct LDI-MS analysis was successfully performed on the resulting covalently bound supported oligomers. To demonstrate the potential application of this class of hybrid bio-organic-inorganic material as supported catalysts, one of the methionine-functionalized OMSs was used to promote disulfide bond formation in a model peptide.

International audience; A wide range of chemical reagents are available to study the protein-protein interactions or protein structures. After reaction with such chemicals, covalently modified proteins are digested, resulting in shorter peptides that are analyzed by mass spectrometry (MS). Used especially when NMR of X-ray data are lacking, this methodology requires the identification of modified species carrying relevant information, among the unmodified peptides. To overcome the drawbacks of existing methods, we propose a more direct strategy relying on the synthesis of solid-supported cleavable monofunctional reagents and cross-linkers that react with proteins and that selectively release, after protein digestion and washings, the modified peptide fragments ready for MS analysis. Using this Solid-Phase Cross-Linking (SPCL) strategy, only modified sequences are analyzed and consistent data can be easily obtained since the signals of interest are not masked or suppressed by over-represented unmodified materials.

International audience; The Pipecolic linker is a new highly versatile handle which immobilizes on solid support through a carboxylic acid function a wide range of amines, alcohols, and hydrazines. The anchoring step on pipecolic resin is very easy and efficient, and compounds are released with high purities upon acidic treatment. During this treatment, an oxazolonium intermediate is hydrolyzed, yielding the cleavage of ester or amide bond and the release of free carboxylic acid of the starting linker. In this study, we report the possibility of recycling the pipecolic resin after the use of several trifluoroacetic acid (TFA) cleavage cocktails. We demonstrate that it can be reused up to five times without significant loading decrease.

International audience

International audience; We report our contribution to the systematic investigation of peptide fragmentations performed on high-performance Tof equipement operating in MS and MS/MS modes, such as ESI-QqTof and MALDI-Tof/Tof intruments that are commonly available today in proteomic laboratories. Whereas the former analyzer's configuration provides low-energy collision-induced dissociations (CID), the latter allows tunable activation methods of the selected parent ion to induce either metastable laser-induced dissociations (LID) or high-energy CID ('gas on spectra LID'). Fragmentation of the monoprotonated ion of 53 peptides (FW 807-2853 g/mol) was undertaken upon low-energy CID on an ESI-QT of mass spectrometer (Waters) as well as high-energy CID and LID conditions on a MALDI Ultraflex mass spectrometer (Bruker). Systematic comparison of MS/MS spectra provided useful information on the performance of each piece of equipment for efficient peptide sequencing and also insights into the observed fragmentation behaviors.

154MF Times Cited:4 Cited References Count:51; Efficient peptide sequencing relies on both high quality MS/MS data acquisition and exhaustive knowledge of gas-phase dissociation mechanisms. We report our contribution to the elaboration of more comprehensive fragmentation models required for efficient automated MS/MS spectra interpretation. Following a statistical approach, various peptides (296 sequences of variable compositions and lengths) were prepared and subjected to low-energy collision-induced dissociations (CID) in an electrospray hybrid instrument (ESI-Q-q-Tof type mass spectrometer) that has retained relatively limited attention so far. Besides, our studies were focused on low molecular weight singly charged peptides that often failed to be identified by sequencing algorithms. Only half of the studied compounds showed charge directed dissociations in accordance with the mobile proton model producing fragment ions directly related to the primary sequence. For the peptides that did not exhibit the expected fragment ion series, alternative dissociation behaviors issued from complex rearrangements were evidenced.

The behavior of C-terminal amidated and carboxylated peptides upon low-energy collision-induced dissociation (CID) was investigated. Two sets of 76 sequences of variable amino acid compositions and lengths were synthesized as model compounds. In most cases, C-terminal amidated peptides were found to produce, upon CID, an abundant loss of ammonia from the protonated molecules. To validate such MS/MS signatures, the studied peptides contained amino acids that can potentially release ammonia from their side chains, such as asparagine, glutamine, tryptophan, lysine and arginine. Arginine, and to a lesser extent lysine, was shown to induce a competitive fragmentation leading to the loss of ammonia from their side chains, thus interfering with the targeted backbone neutral release. However, when arginine or lysine was located at the C-terminal position mimicking a tryptic digest, losses of ammonia from the arginine side chain and from the peptide backbone were completely suppressed. Such results were discussed in the frame of peptidomic or proteomic studies in an attempt to reveal the presence of C-terminal amidated peptides or proteins.

A series of aminotroponimine and aminotropone derivatives were studied by electrospray mass spectrometry. MS and MS/MS data were acquired according to automated procedures. In the case of mono- and di-halogenated compounds, their specific behavior upon collisionally-activated dissociation conditions provided rapid structural assignments through inexpensive isotopic labeling MS/MS experiments.

International audience; Supported peptide and drug-like organic molecule libraries were profiled in single nondestructive imaging static secondary ion mass spectrometric experiments. The selective rupture of the bond linking the compound and the insoluble polymeric support (resin) produced ions that were characteristic of the anchored molecules, thus allowing unambiguous resin bead assignment. Very high sensitivity and specificity were obtained with such a direct analytical method, which avoids the chemical release of the molecules from the support. Libraries issued from either mix-and-split or parallel solid-phase organic syntheses were profiled, demonstrating the usefulness of such a technique for characterization and optimization during combinatorial library development. Moreover, the fact that the control was effected at the bead level whatever the structure and quantity of the anchored molecules allows the sole identification of active beads selected from on-bead screening. Under such circumstances, the time-consuming whole-library characterization could thus be suppressed, enhancing the throughput of the analytical process.

International audience; To evaluate the amount of pyroglutamic acid formation during the synthesis of glutamic acid containing peptides, resin beads were substituted by known amounts of Fmoc-Glu(O(t)Bu)-Phe-Ala-NH2 and the corresponding side- product Pyr-Phe-Ala-NH2 and directly analyzed by time of flight-secondary ion mass spectrometry. Detection and quantification of pyroglutamic acid was achieved without release of the peptide chains in solution.

Some of the chemistry of amino acids going on in our laboratory (Laboratoire des Amino acides Peptides et Protéines) is described as well as some mass spectrometry methodology for their characterization particularly on solid supports. Several aspects are presented including: (i) the stereoselective synthesis of natural and unnatural amino acids using 2-hydroxypinan-3-one as chiral auxiliary; (ii) the stereoselective synthesis of natural and unnatural amino acids by deracemization of alpha-amino acids via their ketene derivatives; (iii) the synthesis of alpha-aryl-alpha-amino acids via reaction of organometallics with a glycine cation; (iv) the diastereoselective synthesis of glycosyl-alpha-amino acids; (v) the synthesis of beta-amino acids using alpha-aminopyrrolidinopiperazinediones as chiral templates; (vi) the reactivity of urethane-N-protected N-carboxyanhydrides. To characterize natural and non natural amino acids through their immonium ions by mass spectrometry, some methodology is also described.

A novel strategy for the synthesis of peptide-bioorganic hybrid silica materials is reported. It relies on the design of hybrid peptides bearing a trialkoxysilane group. These reactive monomers are covalently attached to silica without requiring any prior surface modification. As an example, a bioorganic-inorganic OMS aldolisation catalyst is prepared using the peptide sequence Pro-Pro-Asp-Lys.

International audience; We investigated a variant of desorption/ionization on porous silicon (DIOS) mass spectrometry utilizing an aqueous suspension of either porous silica gel or porous alumina (pore size of 60 and 90 A, respectively). Laser desorption/ionization (LDI) from samples directly deposited on a stainless steel surface without any inorganic substrates was also achieved. Synthetic peptides designed to cover large sequence diversity constituted our model compounds. Sample preparation, including material conditioning, peptide solubilization, and deposition protocol onto standard matrix-assisted laser desorption/ionization (MALDI) probe, as well as ionization source tuning were optimized to perform sensitive reproducible LDI analyses. The addition of either a cationizing agent or an alkali metal scavenger to the sample suspension allowed modification of the ionization output. Comparing hydrophilic silica gel to hydrophobic reversed-phase silica-gel as well as increasing material pore size provided further insights into desorption/ionization processes. Furthermore, mixtures of peptides were analyzed to probe the spectral suppression phenomenon when no interfering organic matrix was present. The results gathered from synthetic peptide cocktails indicated that LDI mass spectrometry on silica gel or alumina constitutes a promising complementary method to MALDI in proteomics for peptide mass fingerprinting.