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In untargeted metabolomics, the unambiguous identification of metabolites remains a major challenge. This requires high-quality spectral libraries for reliable metabolite identification, which is essential for translating metabolomics data into meaningful biological information. Several attempts have been made to generate reproducible product ion spectra (PIS) under a low collision energy ( E ) regime and nonresonant collisional conditions but have not fully succeeded. We examined the ERMS (energy-resolved mass spectrometry) breakdown curves of two lipo-amino acids and showed the possibility to highlight "singular points", called Lab ) regime and nonresonant collisional conditions but have not fully succeeded. We examined the ERMS (energy-resolved mass spectrometry) breakdown curves of two lipo-amino acids and showed the possibility to highlight "singular points", called descriptors hereafter (linked to respective E Lab depending on the instrument), for each of the monomodal product ion profiles. Using several instruments based on different technologies, the PIS recorded at these specific E Lab sites shows remarkable similarities. The descriptors appeared as being independent of the fragmentation mechanisms and can be used to overcome the main instrumental effects that limit the interoperability of spectral libraries. This proof-of-concept study, performed on two particular lipo-amino acids, demonstrates the high potential of ERMS-derived information to determine the instrument-specific E Lab at which PIS recorded in nonresonant conditions become highly similar and instrument-independent, thus comparable across platforms. This innovative but straightforward approach could help remove some of the obstacles to metabolite identification in nontargeted metabolomics, putting an end to a challenging chimera.

There is an urgent need to implement holistic and untargeted doping control protocols with improved discriminatory power, compared to conventional methods that only target doping agents. Metabolomics, which aims to characterize all metabolites present in biological matrices, could fulfill this need. In this context, the aim of this study was to evaluate the impact of environmental factors on the ability to obtain a metabolic signature of stanozolol administration in horse doping situation. Urine samples from 16 horses breeded in two different places were collected over a one-year period, before, during and seven months after the administration of stanozolol, a horse doping agent. Metabolomic analysis was performed using ultra-high pressure reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (MS). Results showed a major impact of the nutritional regimen, drug administration (for de-worming purpose) and breeding place on the metabolite profiles of horse urines, which hampered the detection of metabolic perturbations induced by stanozolol administration. After having used MS/MS experiments to characterize some MS features related to these environmental factors, we showed that highlighting and then removing the features impacted by these confounding factors before performing supervised multivariate statistical analyses could address this issue. In conclusion, adequate consideration should be given to environmental and physiological factors; otherwise, they can emerge as confounding factors and conceal doping administration.

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.

Analytical methods for food control are mainly focused on restricted lists of well-known contaminants. This paper shows that liquid chromatography-high-resolution mass spectrometry (LC/ESI-HRMS) associated with the data mining tools developed for metabolomics can address this issue by enabling (i) targeted analyses of pollutants, (ii) detection of untargeted and unknown xenobiotics, and (iii) detection of metabolites useful for the characterization of food matrices. A proof-of-concept study was performed on 76 honey samples. Targeted analysis indicated that 35 of 83 targeted molecules were detected in the 76 honey samples at concentrations below regulatory limits. Furthermore, untargeted metabolomic-like analyses highlighted 12 chlorinated xenobiotics, 1 of which was detected in lavender honey samples and identified as 2,6-dichlorobenzamide, a metabolite of dichlobenil, a pesticide banned in France since 2010. Lastly, multivariate statistical analyses discriminated honey samples according to their floral origin, and six discriminating metabolites were characterized thanks to the MS/MS experiments.

Control of the ion internal energy in mass spectrometry is needed to establish a workable mass spectral library. The purpose of this study is to understand and to compare the pressure effects on the collision-induced dissociation (CID) spectrum pattern recorded using triple quadrupole instruments. The monoprotonated Leucine enkephalin [YGGFL, H(+)] was used as a thermometer molecule to calibrate the electrospray ionization (ESI) and the CID internal energies deposited on the molecular species and the time scale of ion decompositions. The survival yield and the ratio of a(4)/b(4) fragment ions were mainly monitored. The energy uptake for the ESI source geometry used in our study has no impact on the CID spectrum fingerprint. The collision cell pressure for the [YGGFL, H(+)] has a major influence on the SY curves slope and on the experimental time scale. To demonstrate the pressure effect on internal energy distribution, three models (threshold, thermal and collisional) based on RRKM theory were built using the Masskinetics software. As a result, the limit of each model is discussed, and the investigation demonstrates that the thermal model, using truncated Maxwell-Boltzmann internal energy distribution, is well-suited for simulating the experimental data at high pressure widely used in the analytical conditions., (Copyright © 2013 John Wiley & Sons, Ltd.)

Numerous studies have highlighted the role of the proton donor characteristics of the phenol group of 17β-estradiol (E(2)) in its association with the estrogen receptor alpha (ERα). Since the substitutions at position C((11)) have been reported to modulate this association, we hypothesized that such substitutions may modify the phenol acidity. Hence, phenol gas-phase acidity of nine C((11))-substituted E(2)-derivatives were evaluated using the extended Cooks' kinetic method, which is a method widely used to determine thermochemical properties by mass spectrometry. To enhance accuracy in data collection we recorded data from several instruments, including quadrupole ion trap, triple quadrupole, and hybrid QqTOF. Indeed, we report for the first time the use of the QqTOF instrument to provide a novel means to improve data accuracy by giving access to an intermediate effective temperature range. All experimental gas-phase acidity values were supported by theoretical calculations. Our results confirmed the ability of distant substituents at C((11)) to modulate the phenol acidity through electrostatic interactions, electron withdrawing inductive effects, and mesomeric effects. However, no relationship was found between the phenol gas-phase acidity of investigated steroids and their binding affinity for ERα assessed in solution. Thus, our results highlight that the intrinsic properties of the hormone do not influence sufficiently the stabilization of the hormone/ERα complex. It is more likely that such stabilization would be more related to factors depending on the environment within the binding pocket such as hydrophobic, steric as well as direct intermolecular electrostatic effects between ERα residues and the substituted steroidal estrogens.

Lasso peptides constitute a class of bioactive peptides sharing a knotted structure where the C-terminal tail of the peptide is threaded through and trapped within an N-terminal macrolactam ring. The structural characterization of lasso structures and differentiation from their unthreaded topoisomers is not trivial and generally requires the use of complementary biochemical and spectroscopic methods. Here we investigated two antimicrobial peptides belonging to the class II lasso peptide family and their corresponding unthreaded topoisomers: microcin J25 (MccJ25), which is known to yield two-peptide product ions specific of the lasso structure under collision-induced dissociation (CID), and capistruin, for which CID does not permit to unambiguously assign the lasso structure. The two pairs of topoisomers were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) upon CID, infrared multiple photon dissociation (IRMPD), and electron capture dissociation (ECD). CID and ECD spectra clearly permitted to differentiate MccJ25 from its non-lasso topoisomer MccJ25-Icm, while for capistruin, only ECD was informative and showed different extent of hydrogen migration (formation of c•/z from c/z•) for the threaded and unthreaded topoisomers. The ECD spectra of the triply-charged MccJ25 and MccJ25-lcm showed a series of radical b-type product ions (b'/•(n)). We proposed that these ions are specific of cyclic-branched peptides and result from a dual c/z• and y/b dissociation, in the ring and in the tail, respectively. This work shows the potentiality of ECD for structural characterization of peptide topoisomers, as well as the effect of conformation on hydrogen migration subsequent to electron capture., (© American Society for Mass Spectrometry, 2011)

The metabolome is characterized by a large number of molecules exhibiting a high diversity of chemical structures and abundances, requiring complementary analytical platforms for extensive coverage. Of these analytical platforms, atmospheric pressure ionization Fourier transform mass spectrometry (FT/MS) instruments are popular because they provide accurate mass measurements with ppm and even sub-ppm errors, and also high and ultra-high resolving power. In this article, we evaluate the improvements provided for metabolomics by different types of FT/MS instruments, together with the ability of these platforms to cover the various analytical requirements: global metabolite profiling, absolute quantification and also structural characterization, of metabolomics. The specificities of FT/MS in terms of data pre-processing and the input of accurate mass measurements for biological interpretation and for highlighting metabolic networks are also addressed.

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.

Desorption/ionization on porous silicon (DIOS) is a very useful technique in the case of small molecular weight compounds, compared to the matrix-assisted laser desorption ionization (MALDI). This is because MALDI generates matrix-related ions that overlap with the mass range of interest. The aim of our work was to investigate the suitability of the DIOS technique in the case of fatty acids in negative ion mode. The analysis of the chosen fatty acid models, nonadecanoic acid (C(19)H(38)O(2)) and heneicosanoic acid (C(21)H(42)O(2)), gave rise to the observation of the deprotonated monomeric species and selective cationized multimeric species. This cation selectivity was further elucidated by complementary studies based on the addition of various metals such as Ag(I), Zn(II), Fe(II), and also Cu(II). Specific behavior, depending upon the introduced metal, was highlighted by different redox reaction processes and also metastable decompositions (in PSD mode)., (Copyright 2006 John Wiley & Sons, Ltd.)

The study of low molecular weight compounds by matrix-assisted laser desorption/ionization (MALDI) is difficult because of the presence of ions originating from the matrix in the low-m/z range. In order to resolve these problems, new matrix-free approaches were developed based on laser desorption/ionization from the surface of various materials such as graphite and porous silicon. Our work involves the use of 'desorption ionization on porous silicon mass spectrometry' (DIOS-MS) in the negative ion mode to study fatty acid compounds. The potential of the DIOS-MS technique is shown and an insight into the ionization mechanism provided., (Copyright 2006 John Wiley & Sons, Ltd.)

Hemoglobin-based oxygen therapeutics are prepared by reaction of hemoglobin with cross-linking molecules and are utilized as blood substitutes. They can be used as doping agents to increase the oxygen-carrying capacity of hemoglobin. We have compared a glutaraldehyde-polymerized bovine hemoglobin (Oxyglobin, Biopure Corp.) with natural bovine hemoglobin by mass spectrometry in order to detect specific fragment ions of the cross-linked protein for further potential applications in doping control of human blood samples. HCl acid (6 N) hydrolysis was performed in parallel on both proteins. Hydrolysates were then analyzed by direct infusion electrospray mass spectrometry (ESIMS) using a triple quadrupole mass spectrometer. Confirmation and precision were obtained by LC-ESIMS(n) experiments performed on an ion trap mass spectrometer. Chromatographic and mass spectrometry data allowed detection of two potential Oxyglobin-specific ions--m/z 299 and 399--that were shown to lose a 159 u neutral fragment under collision-induced dissociation conditions. Thus, monitoring of constant neutral loss of 159 u on acid hydrolysates of human serum samples spiked with different amounts of Oxyglobin has proved to be an efficient screening method to specifically detect and identify Oxyglobin. LC-MS of the spiked serum sample hydrolysates enabled detection of Oxyglobin at a detection limit of 4 g x L(-1).

The Maillard reaction is commonly encountered during food processing or storage, and also in human nutrition, hence there is a need for analytical methodologies to identify and characterize the modified proteins. This paper reports specific methods using mass spectrometric techniques to localize protein modifications induced by lactose and galactose on beta-lactoglobulin (beta-Lg) under solid-state glycation conditions. The extent of glycation was first determined by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The specific identification of lactose-modified amino acid residues was realized using both NanoESI-MS, NanoESI-MS/MS (neutral loss scanning modes) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) (with and without guanidination of lysine residues) on unfractionated digests. The results indicated that, after 8.25 h of incubation, the lysine residues were the main targets of lactose-induced modification. In addition to the 15 lysine residues, Leu1 (NH2 terminal) and the Arg124 were also found to be modified, thus leading to a total of 17 different modified amino acid residues (versus 15 found by LC/ESI-MS measurement). In a second set of experiments, different strategies consisting of constant neutral loss and precursor ion scanning were compared to characterize galactose-induced modifications. Owing to the high level of beta-Lg glycation, the combined use of these different strategies appeared to be necessary for determining the galactose-modified sites after 8.25 h of incubation. Thus, among the 22 galactose adducts deduced from the LC/ESI-MS measurement, apart from the N-terminal and classical lysine residues, we also observed a few arginine residues (Arg40, Arg124 and Arg148) that were modified, and also dialkylations on specific lysine residues (Lys47, Lys75)., (Copyright 2003 John Wiley & Sons, Ltd.)

Organic peroxides such as the cumene hydroperoxide I (M(r) = 152 u), the di-tert-butyl peroxide II (M(r) = 146 u) and the tert-butyl peroxybenzoate III (M(r) = 194 u) were analyzed by atmospheric pressure chemical ionization mass spectrometry using a water-methanol mixture as solvent with a low flow-rate of mobile phase and unusual conditions of the source temperature (< or =50 degrees C) and probe temperature (70-200 degrees C). The mass spectra of these compounds show the formation of (i) an [M + H](+) ion (m/z 153) for the hydroperoxide I, (ii) a stable adduct [M + CH(3)OH(2)](+) ion (m/z 179) for the dialkyl peroxide II and (iii) several protonated adduct species such as protonated molecules (m/z 195) and different protonated adduct ions (m/z 227, 389 and 421) for the peroxyester III. Tandem mass spectrometric experiments, exact mass measurements and theoretical calculations were performed for characterize these gas-phase ionic species. Using the double-well energy potential model illustrating a gas-phase bimolecular reaction, three important factors are taken into account to propose a qualitative interpretation of peroxide behavior toward the CH(3)OH(2) (+), i.e. thermochemical parameters (DeltaHdegrees(reaction)) and two kinetic factors such as the capture constant of the initial stable ion-dipole and the magnitude of the rate constant of proton transfer reaction into the loose proton bond cluster., (Copyright 2003 John Wiley & Sons, Ltd.)

The Nobel Prize in Chemistry for 2002 has been awarded to two powerful spectroscopic methodologies through three valorous scientists, John Fenn and Koichi Tanaka, for mass spectrometry and Kurt Wüthrich for nuclear magnetic resonance. These techniques were previously known for their intensive use in chemical analysis. They are now developed at the chemistry/biology interface. Two new methods of soft ionization in mass spectrometry and a strategy of sequential assignment of nuclear magnetic resonance signals of biopolymers now allow the use of these powerful and complementary methodologies for the structural analysis of biological macromolecules, proteins, nucleic acids (DNA, RNA) and polysaccharides. Through the elucidation of their planar and three-dimensional structures and of the molecular mechanisms that govern their interactions, these techniques now may afford precious clues for understanding the molecular mechanisms of life.

The kinetics of the reaction between the octanucleotide d(TTGGCCAA) in the single-stranded form in pure water and the platinum complex [Pt(NH3)3(H2O)]2+ was investigated by electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) mass spectrometries coupled with enzymatic degradation of the adducts. These methods led to the determination of specific rate constants of platination. The global rate constant characteristic of the formation of adducts on each 5'- or 3'-guanine were measured by electrospray ionization analysis. The ratios between the 5'- and 3'-adducts were determined from enzymatic degradation of the final reaction mixture and MALDI analysis. The platination in water is approximately eight times faster than in 0.1 M NaClO4. The selectivity of platination is a factor of 2 in favor of the 5'-guanine, and similar to that observed for the reaction between d(CTGGCTCA) and [Pt(NH3)3(H2O)]2+ in 0.1 M NaClO4.