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Selenium (Se) is an essential element being present in the form of the naturally occurring amino acid selenocysteine (Sec), 25 human proteins involved in different cellular pathways contain Sec. As the most potent intracellular soft Lewis base, selenocysteine (SeCys) is able to bind electron poor soft acids as heavy metals, of awareness for environmental and human toxicology, Hg ions bind Se by means of higher equilibrium constants than sulfur (ca. 106 times), therefore these values compensate the lower cellular abundance (105 times) of selenols compared to thiols[2]. In this communication we present a comparative reactivity study of Hg(I) and Hg(II) compounds with model peptides: vasopressin (AVP) hormone with antidiuretic and vasopressor actions and its Sec containing analogs. These peptides were synthesized either by standard solid phase peptide Fmoc or Boc protocols. The metal ion interaction with these peptides was investigated by RP- LC coupled with electrospray MS/MS detection (LC-MS/MS). We observed mono, bis and bridged peptide metallations as detailed in the Scheme. Taking into consideration the stability of Se-Hg bonds, our results support the hypothesis of a binding preference of Hg to Sec residues in selenoproteins.

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.

As part of the de novo sequencing issue, new approaches have to be found to sequence small natural peptides (

International audience; N-terminal derivatization of proteins with permanently positive charged reagent constitutes a viable mass spectrometry bottom-up strategy for systematic confident identification of protein N-termini. Although shotgun nanoLC-ESI-MS/MS approaches are commonly applied in that context by dissociating multiply charged molecular ions upon low energy collision-induced dissociations (CID), we were keen to investigate an alternative sequencing method reckoning on more comprehensive high energy charge remote fragmentations (CRF) available with a MALDI-Tof/Tof instrument. Hence, two tunable activation methods, i. e. metastable laser-induced dissociations (LID) and high energy CID, were applied to several synthetic N-terminal tris(trimethoxyphenyl)phosphonium (TMPP) acetyl peptides and their MS/MS behaviors were compared with low energy CID MS/MS data (LC-ESI-QqTof equipment). The amino acid composition was found to play a significant role in the peptide backbone dissociation pathways of these fixed singly charged ions. Proline, arginine, aspartic acid, glutamic acid, but also surprisingly aliphatic residues were impacting the expected sequence fragmentation pattern.

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

α-amidation of peptide sequences is a common post-translational modification in the living world. Since the majority of these C-terminal amidated peptides are bioactive, there is hence a great interest to identify and characterize them from biological matrices and natural extracts. Regarding conventional separative methods dedicated to peptides (such as HPLC or CE), elution protocols must be carefully optimized hampering straightforward LC-MS analysis of complex samples. From a mass spectrometry point of view, they are difficult to pinpoint owing to the only 1 Da mass difference between the post-translational amidated and the corresponding native carboxylated forms producing overlapping isotopic contributions of both molecular ions. To circumvent this analytical difficulty, usage of energy-resolved tandem mass spectrometry experiments and of the survival yield technique was investigated. Pair of peptides were thus dissociated in positive and negative mode according to the survival yield technique, in MS

International audience; Cone snails are venomous marine predators that rely on fast-acting venom to subdue their prey and defend against aggressors. The conotoxins produced in the venom gland are small disulfide-rich peptides with high affinity and selectivity for their pharmacological targets. A dominant group comprises α-conotoxins, targeting nicotinic acetylcholine receptors. Here, we report on the synthesis, structure determination and biological activity of a novel α-conotoxin, CIC, found in the predatory venom of the piscivorous species Conus catus and its truncated mutant Δ-CIC. CIC is a 4/7 α-conotoxin with an unusual extended N-terminal tail. High-resolution NMR spectroscopy shows a major influence of the N-terminal tail on the apparent rigidity of the three-dimensional structure of CIC compared to the more flexible Δ-CIC. Surprisingly, this effect on the structure does not alter the biological activity, since both peptides selectively inhibit α3β2 and α6/α3β2β3 nAChRs with almost identical sub- to low micromolar inhibition constants. Our results suggest that the N-terminal part of α-conotoxins can accommodate chemical modifications without affecting their pharmacology.

Objectives Platinum salts are commonly used in hyperthermic intraperitoneal chemotherapy (HIPEC) for digestive tract cancer treatment. During HIPEC with oxaliplatin for peritoneal metastases (PMs) treatment, the ovaries are directly exposed to the drug, questioning about ovarian resection and the potential impact of the drug on ovarian functionality, especially in young women of childbearing age. The goal of this work is to understand unwanted damages to the ovaries during HIPEC therapy by the determination of the concentration and distribution of platinum in ovaries in order to address its potential toxicity. Methods Mass spectrometry imaging techniques, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS), were used to study the penetration of oxaliplatin in ovaries after HIPEC treatment. Results MALDI-MS allowed the localization of an oxaliplatin-derivative (m/z 456.2) at the periphery of the ovaries. The quantitative LA-ICP MS maps confirmed the localization of elemental platinum as well as in the central part of ovaries from patients who received a previous platinum salt-based chemotherapy. Conclusions LA-ICP MS images showed that platinum diffusion was extended in cases of previous systemic treatment, questioning about platinum derivatives gonado-toxicity when combining the two treatments.

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.

International audience; Six Trichosporon asahii strains designated ICVY021, ICVY022, ICVY023, ICVY024, ICVY025 and ICVY026 were isolated from camel milk and studied for their inhibitory activities against a large number of fungi and bacteria as target organisms. Only strain ICVY021 was able to inhibit Kocuria rhizophila CIP 53.45, through production of an extracellular heat-stable, proteinaceous antibacterial peptide, with partial amino acid sequences of PPFPK and CTHV(L/I)(K/Q) or TCHV(L/I)(K/Q), determined using LC/MS/MS. This peptide, named oranicin P16, was thought to impede the cell-division mechanism. The anti-K. rhizophila activity was confirmed, in situ , using skim milk as a food matrix. T. asahii ICVY021 did not show any hemolytic activity or any cytotoxicity effect against eukaryotic Caco-2 cells. T. asahii ICVY021 was sensitive to azoles antifungal compounds, but not to 5-fluorocytosine and anidulafungin.

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.

The asp viper Vipera aspis aspis is a venomous snake found in France, and despite its medical importance, the complete toxin repertoire produced is unknown. Here, we used a venomics approach to decipher the composition of its venom. Transcriptomic analysis revealed 80 venom-annotated sequences grouped into 16 gene families. Among the most represented toxins were snake venom metalloproteases (23%), phospholipases A2 (15%), serine proteases (13%), snake venom metalloprotease inhibitors (13%) and C-type lectins (12%). LC-MS of venoms revealed similar profiles regardless of the method of extraction (milking vs defensive bite). Proteomic analysis validated 57 venom-annotated transcriptomic sequences (>70%), including one for each of the 16 families, but also identified 7 sequences not initially annotated as venom proteins, including a serine protease, a disintegrin, a glutaminyl-peptide cyclotransferase, a proactivator polypeptide-like and 3 aminopeptidases. Interestingly, phospholipases A2 were the dominant proteins in the venom, among which included an ammodytoxin B-like sequence, which may explain the reported neurotoxicity following some asp viper envenomations. In total, 87 sequences were retrieved from the Vipera aspis aspis transcriptome and proteome, constituting a valuable resource that will help in understanding the toxinological basis of clinical signs of envenoming and for the mining of useful pharmacological compounds. Biological significance The asp viper (Vipera aspis aspis) causes several hundred envenomations annually in France, including unusual cases with neurological signs, resulting in one death per year on average. Here, we performed a proteotranscriptomic analysis of V. a. aspis venom in order to provide a better understanding of its venom composition. We found that, as in other Vipera species, phospholipase A2 dominates in the venom, and the presence of a sequence related to ammodytoxin B may explain the reported neurotoxicity following some asp viper envenomations. Thus, this study will help in informing the toxinological basis of clinical signs of envenoming.

Cone snails produce a fast-acting and often paralyzing venom, largely dominated by disulfide-rich conotoxins targeting ion channels. Although disulfide-poor conopeptides are usually minor components of cone snail venoms, their ability to target key membrane receptors such as GPCRs make them highly valuable as drug lead compounds. From the venom gland transcriptome of Conus miliaris, we report here on the discovery and characterization of two conopressins, which are nonapeptide ligands of the vasopressin/oxytocin receptor family. These novel sequence variants show unusual features, including a charge inversion at the critical position 8, with an aspartate instead of a highly conserved lysine or arginine residue. Both the amidated and acid C-terminal analogues were synthesized, followed by pharmacological characterization on human and zebrafish receptors and structural investigation by NMR. Whereas conopressin-M1 showed weak and only partial agonist activity at hV1bR (amidated form only) and ZFV1a1R (both amidated and acid form), both conopressin-M2 analogues acted as full agonists at the ZFV2 receptor with low micromolar affinity. Together with the NMR structures of amidated conopressins-M1, -M2 and -G, this study provides novel structure-activity relationship information that may help in the design of more selective ligands.

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.

International audience; Mass Spectrometry Imaging is an effective technology that allows to determine the in-situ distribution of endogen and/or exogen small molecules. It is a rapidly emerging approach for visualizing drugs and their metabolites within biological tissues. Matrix-Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry Imaging (MSI) coupled to high resolving power analyzer (e.g. TOF) was already investigated for metallodrug localization and metabolization studies, but was proved to suffer from a lack of sensitivity and resolution, leading to poor coverage and assignment. To counter these technological limitations, the use of ultra-high resolving power analyzer such as Fourier Transform Ion Cyclotron Resonance (FTICR) could be revealed as a technique of choice. The high field FTICR MS provides ultra-high resolving power and mass accuracy that allows exhaustive molecule coverage and non-ambiguous molecular formula assignments. Platinum derivatives, such as oxaliplatin, are widely used as therapeutic agents for cancer treatment. The assessment of their intake, distribution and metabolism within the organs is important to know the risks associated with their use. In this study, MALDI FTICR MSI analyses were performed to better understand the penetration and metabolization of platinum derivatives in ovaries of women treated by Hyperthermic Intraperitoneal Chemotherapy (HIPEC) for peritoneal metastasis of colorectal or appendicular origin. Twelve ovary sections, from six ovary samples in six women donors, before and after treatment, were analyzed with 120 μm spatial resolution. For the first time, the high resolving power (220,000 at m/z 457) and sub-ppm accuracy (

Imidazoquinoxaline derivatives (imiqualines) are a new series of anticancer compounds. Two lead compounds (EAPB0203 and EAPB0503) with remarkable in vitro and in vivo activity on melanoma and T-cell lymphomas have been previously identified. The modulation of the chemical structure of the most active compound, EAPB0503, has led to the synthesis of two compounds, EAPB02302 and EAPB02303, 7 and 40 times more active than EAPB0503 against A375 human melanoma cancer cell line, respectively. The aim of this study was to develop and validate a sensitive and accurate liquid chromatography-electrospray ionization-tandem mass spectrometry method to simultaneously quantify EAPB02303 and its potential active metabolite, EAPB02302, in rat and mouse plasma. Analytes were detected in multiple reaction monitoring acquisition mode using an electrospray ionization detector in positive ion mode. Following a liquid-liquid extraction with ethyl acetate, analytes and internal standard were separated by HPLC reversed-phase on a C18 RP18 Nucleoshell column (2.7μm, 4.6×100mm). The method was validated according to FDA and EMA Bioanalytical Method Validation guidelines. The robustness of the method was assessed by introducing small variations in nine nominal analytical parameters. Statistical interpretation was performed by mean of the Student's t-test. Standard curves were generated via unweighted quadratic regression of calibrators (EAPB02303: 1.95-1000ng/mL, EAPB02302: 7.81-1000ng/mL in rat plasma; EAPB02303: 0.98-1000ng/mL, EAPB02302: 1.95-1000ng/mL in mouse plasma). From the analysis of QC samples, intra- and inter-assay precision and accuracy studies demonstrated %R.S.Ds.12.5% and percent deviation from nominal concentration7%. Matrix effects (mean matrix factors from 91.8-108.5% in rat plasma; and from 90.4-102.4% in mouse plasma) and stability assays (recoveries87%) were acceptable and in accordance with the guidelines. No quantifiable carryover effect was observed. The LLOQs were 1.95ng/mL for EAPB02303 and 7.81ng/mL for EAPB02302 in rat plasma, and 0.98ng/mL and 1.95ng/mL for the two compounds in mouse plasma, respectively. This method was successfully implemented to support a mouse pharmacokinetic study following a single intraperitoneal administration of EAPB02303 in male C57Bl/6 mice. The obtained pharmacokinetic parameters of EAPB02303 would be useful to optimize the dosing and the rhythm of administration for subsequent preclinical in vivo activity studies.

Both amide bond protonation triggering peptide fragmentations and the controversial bTo 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 bWhereas it is very difficult to differentiate among protonated oxazolone, diketopiperazine, or lactam bIn contrast to glycine-histidine-containing b

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.

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-MS

Cone snails produce a fast-acting and often paralyzing venom that is usually injected into their prey or predator through a hypodermic needle-like modified radula tooth. Many diverse compounds are found in their venom including small molecules, peptides and enzymes. However, peptidic toxins called conotoxins (10&ndash, 40 residues and 2&ndash, 4 disulfide bonds) largely dominate these cocktails. These disulfide rich toxins are very valuable pharmacological tools for investigating the function of ions channels, G-protein coupled receptors, transporters and enzymes. Here, we report on the synthesis, structure determination and biological activities of two &alpha, conotoxins, CIA and CIB, found in the predatory venom of the piscivorous species Conus catus. CIA is a typical 3/5 &alpha, conotoxin that blocks the rat muscle type nAChR with an IC50 of 5.7 nM. Interestingly, CIA also inhibits the neuronal rat nAChR subtype &alpha, 3&beta, 2 with an IC50 of 2.06 &mu, M. CIB is a 4/7 &alpha, conotoxin that blocks rat neuronal nAChR subtypes, including &alpha, 2 (IC50 = 128.9 nM) and &alpha, 7 (IC50 = 1.51 &mu, M). High resolution NMR structures revealed typical &alpha, conotoxin folds for both peptides. We also investigated the in vivo effects of these toxins on fish, since both peptides were identified in the predatory venom of C. catus. Consistent with their pharmacology, CIA was highly paralytic to zebrafish (ED50 = 110 &mu, g/kg), whereas CIB did not affect the mobility of the fish. In conclusion, CIA likely participates in prey capture through muscle paralysis, while the putative ecological role of CIB remains to be elucidated.

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.

We describe a new class of silicone-containing peptide polymers obtained by a straightforward polymerization in water using tailored chlorodimethylsilyl peptide blocks as monomeric units. This general strategy is applicable to any type of peptide sequences, yielding linear or branched polymer chains composed of well-defined peptide sequences.

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.

When synthesizing arylpiperazine library modified with N-acylated amino acid derivatives (e.g., cyclized aspartic acid, cyclized glutamic acid, proline) we wished to rapidly determine the way of cyclization of N-acylated glutamic acid derivatives. During concomitant cleavage and cyclization two alternative routes were possible—either formation of six-member imide (glutarimide) or five-member lactam. Application of MS/MS and 1H NMR method allowed us to establish that cyclization of N-acylated glutamic acid derivatives preceded to lactams—N-acylated pyroglutamic acid derivatives. Under acidic treatment the Lantern bound N-acylated glutamic acid derivatives underwent concomitant cleavage and cyclization. Herein, we studied two alternative cyclization routes—imides, lactams. It was found that cyclization of N-acylated glutamic acid derivatives yielded pyroglutamic acid derivatives (Structure C).

Objectif Demontrer le potentiel du MALDI couple a la spectrometrie de masse (MS, MS2 et MS3) et a l’imagerie pour distinguer dans le cheveu trois isomeres de la serie JWH (JWH-007, JWH-019 et JWH-122) divergeant uniquement par la position d’un groupe methyl. Methodes Les solutions standard de JWH-007, JWH-019 et JWH-122 ont ete analysees en MS, MS2 et MS3 par MALDI QIT-TOF (Axima Resonance, Shimadzu) et en MS et MS2 par MALDI-7090™ TOF-TOF (Shimadzu) dans un domaine de masse compris entre 50 et 500 Da. Les ions fragments ont ete obtenus selon des processus de fragmentation de basse energie pour le QIT-TOF et de haute energie pour le TOF-TOF. Dans un deuxieme temps, deux cheveux non segmentes ont ete incubes 12 h dans une solution contenant les 3 standards, puis fixes sur une lame de verre conductrice sur laquelle a ensuite ete deposee la matrice acide α-cyano-4-hydroxycinnamique (CHCA). Les cheveux ont ete analyses selon les memes procedures que les standards. Ces analyses ont ete completees par imagerie MALDI avec le QIT-TOF. Les spectres, obtenus avec un pas d’acquisition de 80 μm, ont ete retraites par le logiciel BioMap® pour fournir une cartographie des ions d’interet. Les spectres de masse des solutions standard obtenus a partir du MALDI QIT-TOF et du MALDI-TOF-TOF montrent un ion precurseur commun (m/z 356,2) sans qu’il soit possible de distinguer les 3 isomeres. En MS2, les deux techniques ont permis d’obtenir les ions fils caracteristiques du JWH-122 (m/z 169,1 et 214,1), mais pas ceux des deux autres isomeres d’interet, la fragmentation du JWH-007 et du JWH-019 menant a deux spectres identiques (pics majoritaires : m/z 155,1 et 228,1). Seule la fragmentation en MS3 par QIT-TOF du JWH-007 et du JWH-019 permet d’identifier un ion fragment unique a m/z 158,1 pour le JWH-007, et quatre ions m/z a 144,1 (pic principal), 116,1, 130,1, et 158,1 pour le JWH-019. L’analyse des cheveux selon les memes procedures a donne des resultats comparables. L’imagerie obtenue par le QIT-TOF sur les deux cheveux permet de suivre l’intensite du signal des ions fils le long du cheveu et de distinguer les 3 cannabinoides de synthese ( Annexe A ). Conclusions Avec une preparation simple et rapide, une resolution spatiale elevee et une fragmentation MS3, le couplage imagerie MALDI-MS ouvre de nouvelles perspectives pour caracteriser dans les milieux biologiques des molecules de structure chimique tres voisine. L’application a des cas reels constitue l’etape suivante de ce travail.