Jean-Claude Tabet

Siderophore peptide, a new type of post-translationally modified antibacterial peptide with potent activity

Microcin E492 (MccE492, 7886 Da), the 84-amino acid antimicrobial peptide from Klebsiella pneumoniae, was purified in a post-translationally modified form, MccE492m (8717 Da), from culture supernatants of either the recombinant Escherichia coli VCS257 strain harboring the pJAM229 plasmid or the K. pneumoniae RYC492 strain. Chymotrypsin digestion of MccE492m led to the MccE492m-(74–84) C-terminal fragment that carries the modification and that was analyzed by mass spectrometry and nuclear magnetic resonance at natural abundance. The 831-Da post-translational modification consists of a trimer of N-(2,3-dihydroxybenzoyl)-l-serine linked via a C-glycosidic linkage to a β-d-glucose moiety, itself linked to the MccE492m Ser-84-carboxyl through an O-glycosidic bond. This modification, which mimics a catechol-type siderophore, was shown to bind ferric ions by analysis of the collision-induced dissociation pattern obtained for MccE492m-(74–84) by electrospray ion trap mass spectrometry experiments in the presence of FeCl3. By using a series of wild-type and mutant isogenic strains, the three catechol-type siderophore receptors Fiu, Cir, and FepA were shown to be responsible for the recognition of MccE492m at the outer membrane of sensitive bacteria. Because MccE492m shows a broader spectrum of antibacterial activity and is more potent than MccE492, we propose that by increasing the microcin/receptor affinity, the modification leads to a better recognition and subsequently to a higher antimicrobial activity of the microcin. Therefore, MccE492m is the first member of a new class of antimicrobial peptides carrying a siderophore-like post-translational modification and showing potent activity, which we term siderophore-peptides.

Zinc binding properties of the amyloid fragment Aβ(1–16) studied by electrospray-ionization mass spectrometry

A major hallmark of Alzheimer’s disease (AD) is the strong accumulation in brain of senile plaques, mainly composed of the amyloid-β peptide (Aβ). Recent studies have suggested that the zinc cation would be a possible key mediating factor for the formation of amyloid extracellular deposits, by binding to Aβ and triggering the involved aggregation process. From a previous circular dichroism (CD) study, we have proposed the N-terminal 1–16 region of Aβ(1–16), as the minimal fragment able to specifically bind zinc. Here we investigate the Zn2+ binding properties of Aβ(1–16) by electrospray-ionization mass spectrometry (ESI-MS). The stoichiometry of Aβ(1–16)/Zn2+ association and the relative affinity of different cations towards Aβ(1–16) are investigated by analyzing the mass spectra of Aβ(1–16) in the presence of different cations, introduced alone or in competition. Zn2+ binding sites are determined from collision-induced dissociation (CID) experiments conducted on the Aβ(1–16) cationized species. From these data, Aβ(1–16) is shown to form a 1:1 complex with Zn2+ and to bind up to three cations upon increasing the Zn2+ concentration. Under CID, zinc binding induces specific cleavages after the three histidines of the Aβ(1–16) sequence (H6, H13 and H14), showing their simultaneous implication in the Zn2+ coordination sphere. The binding of Aβ(1–16) to several Zn2+ cations appears less specific, but still implicates the three histidines, each of them behaving thus as an autonomous binding site. A model is proposed to explain both the specific and the aspecific interactions of Zn2+ with Aβ(1–16) that is confirmed here to behave as the minimal zinc-binding region of Aβ.

Hyphenation of atmospheric pressure chemical ionisation mass spectrometry to supercritical fluid chromatography for polar car lubricant additives analysis

Car lubricant additives are added to mineral or synthetic base stocks to improve viscosity and resistance to oxidation of the lubricant and to limit wear of engines. As they belong to various chemical classes and are added to a very complex medium, the base stock, their detailed chromatographic analysis is very difficult and time consuming. In a previous paper, it was demonstrated that supercritical fluid chromatography (SFC) allows the elution of common low-molecular-weight additives. Since their total resolution could not be achieved owing to the limited peak capacity of packed columns, the hyphenation of selective and informative detection methods such as atomic emission detection (AED) was required. Further to results obtained in SFC-AED, this work describes the hyphenation of SFC to atmospheric pressure chemical ionisation ion trap mass spectrometry (MS). SFC-MS hyphenation is detailed: temperature, flow rates of gas and mobile phase introduced in the source, position of the restrictor, ionisation additives and conditions of autotune are studied. Car lubricant monitoring requires negative and positive ionisation modes with or without the addition of ionisation auxiliary solvent according to the nature of additives. Moreover, when sensitivity is of major concern for a selected additive, the autotuning routine of the MS has to be performed in conditions as close as possible to analytical conditions, i.e. under subcritical conditions. Unambiguous identification and structure elucidation of several additives in formulated car lubricants are also presented.

Identification of the products of reaction between pyridoxal phosphate and amiclenomycin and other related 1-amino-cyclohexa-2,5-dienes

Abstract Amiclenomycin, a natural product containing the 1-amino cyclohexa-2,5-diene moiety is an inhibitor of 7;8-diaminopelargonic acid aminotransferase, a pyridoxal phosphate (PLP) dependent enzyme involved in biotin biosynthesis. The postulated mechanism implies the aromatisation of the Schiff base formed between PLP and amiclenomycin. Aromatic adducts have been obtained by heating PLP with amiclenomycin and other related 1-amino cyclohexa-2,5-dienes. They were fully characterized by UV–visible and ESI mass spectrometry and provide standards for identification of the enzyme-derived products.

Ion/molecule reactions with dimethyl ether and dimethyl-d6 ether: differentiation of four isomers contained in patchouli essential oil

In this study, we show that it is possible to differentiate four sesquiterpene isomers (C(15)H(24)) preliminarily separated by gas chromatography/mass spectrometry (GC/MS). Dimethyl ether is evaluated as a selective ionization reagent and the relative abundance of adducts formed with this reagent gas under positive chemical ionization conditions are compared and adduct ions are characterized using collision-induced dissociation. The mechanisms have been confirmed by achieving the same experiments with deuterated dimethyl ether. Copyright 2000 John Wiley & Sons, Ltd.

Identification of an analgesic lipopeptide produced by the probiotic Escherichia coli strain Nissle 1917

Administration of the probiotic Escherichia coli strain Nissle 1917 (EcN) decreases visceral pain associated with irritable bowel syndrome. Mutation of clbA, a gene involved in the biosynthesis of secondary metabolites, including colibactin, was previously shown to abrogate EcN probiotic activity. Here, we show that EcN, but not an isogenic clbA mutant, produces an analgesic lipopeptide. We characterize lipoamino acids and lipopeptides produced by EcN but not by the mutant by online liquid chromatography mass spectrometry. One of these lipopeptides, C12AsnGABAOH, is able to cross the epithelial barrier and to inhibit calcium flux induced by nociceptor activation in sensory neurons via the GABAB receptor. C12AsnGABAOH inhibits visceral hypersensitivity induced by nociceptor activation in mice. Thus, EcN produces a visceral analgesic, which could be the basis for the development of new visceral pain therapies.Escherichia coli Nissle is a probiotic that decreases visceral pain associated with irritable bowel syndrome. Here, the authors show that the microbe produces an analgesic lipopeptide, structurally related to GABA, that can cross the gut epithelial barrier and inhibits visceral hypersensitivity in mice.

Mechanistic study of competitive releases of H2O, NH3 and CO2 from deprotonated aspartic and glutamic acids: Role of conformation

The aims of this study were to highlight the impact of minor structural differences (e.g. an aminoacid side chain enlargement by one methylene group), on ion dissociation under collision-induced dissociation conditions, and to determine the underlying chemical mechanisms. Therefore, we compared fragmentations of deprotonated aspartic and glutamic acids generated in negative electrospray ionization. Energy-resolved mass spectrometry breakdown curves were recorded and MS3 experiments performed on an Orbitrap Fusion for high-resolution and high-mass accuracy measurements. Activated fragmentations were performed using both the resonant and non-resonant excitation modes (i.e., CID and HCD, respectively) in order to get complementary information on the competitive and consecutive dissociative pathways. These experiments showed a specific loss of ammonia from the activated aspartate but not from the activated glutamate. We mainly focused on this specific observed loss from aspartate. Two different mechanisms based on intramolecular reactions (similar to those occurring in organic chemistry) were proposed, such as intramolecular elimination (i.e. Ei-like) and nucleophilic substitution (i.e. SNi-like) reactions, respectively, yielding anions as fumarate and α lactone from a particular conformation with the lowest steric hindrance (i.e. with antiperiplanar carboxyl groups). The detected deaminated aspartate anion can then release CO2 as observed in the MS3 experimental spectra. However, quantum calculations did not indicate the formation of such a deaminated aspartate product ion without loss of carbon dioxide. Actually, calculations displayed the double neutral (NH3+CO2) loss as a concomitant pathway (from a particular conformation) with relative high activation energy instead of a consecutive process. This disagreement is apparent since the concomitant pathway may be changed into consecutive dissociations according to the collision energy i.e., at higher collision energy and at lower excitation conditions, respectively. The latter takes place by stabilization of the deaminated aspartate solvated with two residual molecules of water (present in the collision cell). This desolvated anion formed is an α lactone substituted by a methylene carboxylate group. The vibrational excitation acquired by [(D-H)-NH3]-during its isolation is enough to allow its prompt decarboxylation with a barrier lower than 8.4kJ/mol. In addition, study of glutamic acid-like diastereomers constituted by a cyclopropane, hindering any side chain rotation, confirms the impact of the three-dimensional geometry on fragmentation pathways. A significant specific loss of water is only observed for one of these diastereomers. Other experiments, such as stable isotope labeling, need to be performed to elucidate all the observed losses from activated aspartate and glutamate anions. These first mechanistic interpretations enhance understanding of this dissociative pathway and underline the necessity of studying fragmentation of a large number of various compounds to implement properly new algorithms for de novo elucidation of unknown metabolites.

Evaluation of the High-Field Orbitrap Fusion for Compound Annotation in Metabolomics

Annotation of signals of interest represents a key point in mass spectrometry-based metabolomics studies. The first level of investigation is the elemental composition, which can be deduced from accurately measured masses and isotope patterns. However, accuracy of these two parameters remains to be evaluated on last generation mass spectrometers to determine the level of confidence that can be used during the annotation process. In this context, we evaluated the performance of the Orbitrap Fusion mass spectrometer for the first time and demonstrated huge potential for metabolite annotation via elemental composition determination. This work was performed using a set of 50 standard compounds analyzed under LC/MS conditions in solvent and biological media. Accurate control of the number of trapped ions proved mandatory to avoid space charge effects, ensure sub-ppm mass accuracy (using external calibration), and reliable measurement of isotopic patterns at 500,000 resolution. On the basis of the results, we propose standard optimized experimental conditions for performing robust and accurate untargeted metabolomics on the Orbitrap Fusion at high mass measurement and mass spectral accuracy.

Mechanisms governing the fragmentation of glycerophospholipids containing choline and ethanolamine polar head groups

Glycerophospholipids are the major amphiphilic molecules found in the plasma membrane bilayer of all vertebrate cells. Involved in many biological processes, their huge structural diversity and large concentration scale make their thorough characterization extremely difficult in complex biological matrices. Mass spectrometry techniques are now recognized as being among the most powerful methods for the sensitive and comprehensive characterization of lipids. Depending on the experimental conditions used during electrospray ionization mass spectrometry experiments, glycerophospholipids can be detected as different molecular species (e.g. protonated, sodiated species) when analyzed either in positive or negative ionization modes or by direct introduction or hyphenated mass spectrometry-based methods. The observed ionized forms are characteristic of the corresponding phospholipid structures, and their formation is highly influenced by the polar head group. Although the fragmentation behavior of each phospholipid class has already been widely studied under low collision energy, there are no established rules based on charge-induced dissociation mechanisms for explaining the generation of fragment ions. In the present paper, we emphasize the crucial roles played by ion–dipole complexes and salt bridges within charge-induced dissociation processes. Under these conditions, we were able to readily explain almost all the fragment ions obtained under low-energy collision-induced dissociation for particular glycerophospholipids and lysoglycerophospholipids species including glycerophosphatidylcholines and glycerophosphatidylethanolamines. Thus, in addition to providing a basis for a better comprehension of phospholipid fragmentation processes, our work also highlighted some potentially new relevant diagnostic ions to signal the presence of particular lipid species.

Evaluation and validation of an analytical approach for high-throughput metabolomic fingerprinting using direct introduction–high-resolution mass spectrometry: Applicability to classification of urine of scrapie-infected ewes

Direct injection–mass spectrometry can be used to perform high-throughput metabolomic fingerprinting. This work aims to evaluate a global analytical workflow in terms of sample preparation (urine sample dilution), high-resolution detection (quality of generated data based on criteria such as mass measurement accuracy and detection sensitivity) and data analysis using dedicated bioinformatics tools. Investigation was performed on a large number of biological samples collected from sheep infected or not with scrapie. Direct injection–mass spectrometry approach is usually affected by matrix effects, eventually hampering detection of some relevant biomarkers. Reference compounds were spiked in biological samples to help evaluate the quality of direct injection–mass spectrometry data produced by Fourier Transform mass spectrometry. Despite the potential of high-resolution detection, some drawbacks still remain. The most critical is the presence of matrix effects, which could be minimized by optimizing the sample dilution factor. The data quality in terms of mass measurement accuracy and reproducible intensity was evaluated. Good repeatability was obtained for the chosen dilution factor (i.e., 2000). More than 150 analyses were performed in less than 16 hours using the optimized direct injection–mass spectrometry approach. Discrimination of different status of sheeps in relation to scrapie infection (i.e., scrapie-affected, preclinical scrapie or healthy) was obtained from the application of Shrinkage Discriminant Analysis to the direct injection–mass spectrometry data. The most relevant variables related to this discrimination were selected and annotated. This study demonstrated that the choice of appropriated dilution faction is indispensable for producing quality and informative direct injection–mass spectrometry data. Successful application of direct injection–mass spectrometry approach for high throughput analysis of a large number of biological samples constitutes the proof of the concept.