Spatialized Metabolomic Annotation Combining MALDI Imaging and Molecular Networks.

MALDI mass spectrometry imaging has gained major interest in the field of chemical imaging. This technique makes it possible to locate tens to hundreds of ionic signals on the sample surface without any a priori. One of the current challenges is still the limited ability to annotate signals in order to convert m/z values into probable chemical structures. At the same time, data obtained by LC-MS/MS have benefited from the development of numerous chemoinformatics tools, in particular molecular networks, for their efficient annotation. For the first time, we present here the combination of MALDI-FT-ICR imaging with molecular networks from MALDI-MS/MS data directly acquired on plant tissue sections. Annotation improvements are demonstrated, paving the way for new annotation pipelines for MALDI imaging.

Fourier transform ion cyclotron resonance mass spectrometry at the true cyclotron frequency.

Ion cyclotron resonance (ICR) cells provide stability and coherence of ion oscillations in crossed electric and magnetic fields over extended periods of time. Using the Fourier transform enables precise measurements of ion oscillation frequencies. These precisely measured frequencies are converted into highly accurate mass-to-charge ratios of the analyte ions by calibration procedures. In terms of resolution and mass accuracy, Fourier transform ICR mass spectrometry (FT-ICR MS) offers the highest performance of any MS technology. This is reflected in its wide range of applications. However, in the most challenging MS application, for example, imaging, enhancements in the mass accuracy of fluctuating ion fluxes are required to continue advancing the field. One approach is to shift the ion signal power into the peak corresponding to the true cyclotron frequency instead of the reduced cyclotron frequency peak. The benefits of measuring the true cyclotron frequency include increased tolerance to electric fields within the ICR cell, which enhances frequency measurement precision. As a result, many attempts to implement this mode of FT-ICR MS operation have occurred. Examples of true cyclotron frequency measurements include detection of magnetron inter-harmonics of the reduced cyclotron frequency (i.e., the sidebands), trapping field-free (i.e., screened) ICR cells, and hyperbolic ICR cells with quadrupolar ion detection. More recently, ICR cells with spatially distributed ion clouds have demonstrated attractive performance characteristics for true cyclotron frequency ion detection. Here, we review the corresponding developments in FT-ICR MS over the past 40 years.

How Well Do We Handle the Sample Preparation, FT-ICR Mass Spectrometry Analysis, and Data Treatment of Atmospheric Waters?

FT-ICR MS (Fourier-transform ion cyclotron resonance mass spectrometry) analysis has shown great potential to aid in the understanding of the extremely high molecular diversity of cloud water samples. The main goal of this work was to determine the differences in terms of formula assignment for analytical (i.e., measurement replicates) and experimental replicates of a given cloud water sample. The experimental replicates, obtained by solid phase extraction, were also compared to the results obtained for freeze-dried samples to evaluate whether the presence of salts interferes with the analysis. Two S/N ratios, generally adopted for atmospheric samples, were evaluated, and three different algorithms were used for assignment: DataAnalysis 5.3 (Bruker), Composer (Sierra Analytics), and MFAssignR (Chemical Advanced Resolution Methods Lab). In contrast to other works, we wanted to treat this comparison from the point of view of users, who usually must deal with a simple list of m/z ratios and intensity with limited access to the mass spectrum characteristics. The aim of this study was to establish a methodology for the treatment of atmospheric aqueous samples in light of the comparison of three different software programs, to enhance the possibility of data comparison within samples.

Chemical Processes Involving 18-Crown-6-Ether in Activated Noncovalent Complexes with Protonated Peptides.

These last decades, it has been widely assumed that 18-crown-6-ether (CE) plays a spectator role during the chemical processes occurring in isolated host-guest complexes between peptides or proteins and CE after activation in mass spectrometers. Our present experimental and theoretical results challenge this hypothesis by showing that CE can abstract a proton or a protonated molecule from protonated peptides after activation by collisions in argon or electron capture/transfer. Furthermore, thanks to comparison between experimental and calculated values of collision cross-sections, we demonstrate that CE can change binding site after electron transfer. We also propose detailed mechanisms for these processes.

SPIX: A new software package to reveal chemical reactions at trace amounts in very complex mixtures from high-resolution mass spectra dataset.

RATIONALE: High-resolution mass spectrometry based non-targeted screening has a huge potential for applications in environmental sciences, engineering and regulation. However, it produces large datasets for which full appropriate processing is a real challenge; the development of processing software is the last building-block to enable large-scale use of this approach. METHODS: A new software application, SPIX, has been developed to extract relevant information from high-resolution mass spectral datasets. Dealing with intrinsic sample variability and reducing operator subjectivity, it opens up opportunities and promising prospects in many areas of analytical chemistry. SPIX is freely available at: http://spix.webpopix.org. RESULTS: Two features of the software are presented in the field of environmental analysis. An example illustrates how SPIX reveals photodegradation reactions in wastewater by fitting kinetic models to significant changes in ion abundance over time. A second example shows the ability of SPIX to detect photoproducts at trace amounts in river water, through comparison of datasets from samples taken before and after irradiation. CONCLUSIONS: SPIX has shown its ability to reveal relevant modifications between two series of large datasets, allowing, for instance, the study of the consequences of a given event on a complex substrate. Most of all - and it is to our knowledge the only software currently available allowing this - it can reveal and monitor any kind of reaction in all types of mixture.

Infrared Spectra of Deprotonated Dicarboxylic Acids: IRMPD Spectroscopy and Empirical Valence-Bond Modeling.

Experimental infrared multiple-photon dissociation (IRMPD) spectra recorded for a series of deprotonated dicarboxylic acids, HO2 (CH2 )n CO 2 - (n=2-4), are interpreted using a variety of computational methods. The broad bands centered near 1600 cm-1 can be reproduced neither by static vibrational calculations based on quantum chemistry nor by a dynamical description of individual structures using the many-body polarizable AMOEBA force field, strongly suggesting that these molecules experience dynamical proton sharing between the two carboxylic ends. To confirm this assumption, AMOEBA was combined with a two-state empirical valence-bond (EVB) model to allow for proton transfer in classical molecular dynamics simulations. Upon suitable parametrization based on ab initio reference data, the EVB-AMOEBA model satisfactorily reproduces the experimental infrared spectra, and the finite temperature dynamics reveals a significant amount of proton sharing in such systems.

Spectroscopy and Photodissociation of the Perfluorooctanoate Anion.

Perfluorocarbons, a class of fully fluorinated compounds, are highly persistent and toxic pollutants that are receiving increasing attention due to their widespread environmental distribution. In this study, attention was focused on one compound in particular, namely, perfluorooctanoic acid (PFOA). The first investigation of the UV/VUV photochemistry of the PFOA anion in the gas phase by action spectroscopy of selected ions is reported. Two main relaxation channels were identified: photodissociation and photodetachment. Absolute cross sections for the individual observed processes were measured. DFT calculations and natural transition orbital analysis were carried out to help in the interpretation of the experimental results.

Ultraviolet-visible phototransformation of dehydroacetic acid - Structural characterization of photoproducts and global ecotoxicity.

RATIONALE: The present work is devoted to the structural elucidation of by-products issued from the direct ultraviolet-visible (UV-vis) irradiation of dehydroacetic acid (DHA) in solution and in cosmetic emulsion. METHODS: Analyses were carried out using gas chromatography coupled with ion trap mass spectrometry and by high-performance liquid chromatography coupled with ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (LC/UHRMS). The potential toxicities of by-products were estimated by in silico calculations based on a QSAR (Quantitative Structure-Activity Relationship) approach and by in vitro bioassays conducted on Vibrio fischeri bacteria. RESULTS: Three photoproducts were detected by LC/MS while one photoproduct was detected by GC/MS. The first photoproduct (PP1) corresponds to an isomer of DHA while two isomeric compounds correspond to dimeric structures. The oral rat LD50 of PP1 was evaluated to be 4.5 times lower than that of the parent molecule which classes it in the category 'moderately toxic' on the Hodge and Sterne toxicity classification. In vitro assays on Vibrio fischeri bacteria showed that the global ecotoxicity of the DHA solution increases with irradiation time. CONCLUSIONS: With the exception of one photoproduct, the structures proposed for the photoproducts on the basis of mass spectral interpretation have not been reported in previous studies. All photoproducts, with the exception of dimers, were detected after irradiation in the cosmetic emulsion. This result shows that personal care products containing DHA must be protected from direct sunlight to prevent photodegradation.

Molecular Characterization of Cloud Water Samples Collected at the Puy de Dôme (France) by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Cloud droplets contain dynamic and complex pools of highly heterogeneous organic matter, resulting from the dissolution of both water-soluble organic carbon in atmospheric aerosol particles and gas-phase soluble species, and are constantly impacted by chemical, photochemical, and biological transformations. Cloud samples from two summer events, characterized by different air masses and physicochemical properties, were collected at the Puy de Dôme station in France, concentrated on a strata-X solid-phase extraction cartridge and directly infused using electrospray ionization in the negative mode coupled with ultrahigh-resolution mass spectrometry. A significantly higher number (n = 5258) of monoisotopic molecular formulas, assigned to CHO, CHNO, CHSO, and CHNSO, were identified in the cloud sample whose air mass had passed over the highly urbanized Paris region (J1) compared to the cloud sample whose air mass had passed over remote areas (n = 2896; J2). Van Krevelen diagrams revealed that lignins/CRAM-like, aliphatics/proteins-like, and lipids-like compounds were the most abundant classes in both samples. Comparison of our results with previously published data sets on atmospheric aqueous media indicated that the average O/C ratios reported in this work (0.37) are similar to those reported for fog water and for biogenic aerosols but are lower than the values measured for aerosols sampled in the atmosphere and for aerosols produced artificially in environmental chambers.

Improved Infrared Spectra Prediction by DFT from a New Experimental Database.

This work aims to improve the computation of infrared spectra of gas-phase cations using DFT methods. Experimental infrared multiple photon dissociation (IRMPD) spectra for ten Zn and Ru organometallic complexes have been used to provide reference data for 64 vibrational modes in the 900-2000 cm-1 range. The accuracy of the IR vibrational frequencies predicted for these bands has been assessed over five DFT functionals and three basis sets. The functionals include the popular B3LYP and M06-2X hybrids and the range-separated hybrids (RSH) CAM-B3LYP, LC-BLYP, and ωB97X-D. B3LYP gives the best mean absolute error (MAE) and root-mean-square error (RMSE) values of 7.1 and 9.6 cm-1 , whilst the best RSH functional, ωB97X-D, gives 12.8 and 16.6 cm-1 , respectively. Using linear correlations instead of scaling factors improves the prediction accuracy significantly for all functionals. Experimental and computed spectra for a single complex can show significant differences even when the molecular structure is calculated correctly, and a means of defining confidence limits for any given computed structure is also provided.

Photodegradation of cyprodinil under UV-visible irradiation - chemical and toxicological approaches.

RATIONALE: Cyprodinil is a fungicide active on grapes, strawberries, tomatoes, and many other fruits. Under UV-visible irradiation, it undergoes photodegradation through various processes to form transformation products (TPs) whose structures and potential toxicities are unknown. The structures of the TPs were elucidated by comparing the photodegradation of cyprodinil and cyprodinil-D5 . The potential toxicities of these compounds were compared with that of cyprodinil. METHODS: Aqueous solutions of cyprodinil were irradiated in a reactor equipped with a mercury vapor lamp. Analyses were carried out using high-performance liquid chromatography coupled to a quadrupole time-of-flight (QTOF) mass spectrometer or to a SolarixXR 9.4 Tesla Fourier transform (FT) mass spectrometer. High-resolution mass measurements, MS/MS and isotopic labeling experiments allowed structural elucidation of the cyprodinil TPs. The toxicities were estimated by three tests in silico using the TEST software and in vitro bioassays using Vibrio fischeri bacteria. These bioassays were carried out on irradiated solution for several exposure times and non-irradiated solutions. RESULTS: The structures of 19 photoproducts were characterized by LC/HRMS/MS after 4 h of irradiation of a cyprodinil aqueous solution. The use of cyprodinil-D5 allowed the TPs to be characterized with more confidence. Knowing the structure of the TPs allows the estimation of their potential toxicities by in silico tests. Most of the photoproducts are potentially more toxic than the parent compound, based on the oral rat LD50 values, and most of them might induce more developmental and mutagenic toxicities. In vitro assays on Vibrio fischeri bacteria showed that the global ecotoxicity of the cyprodinil solution significantly increases with irradiation time. CONCLUSIONS: Structures of photoproducts were characterized after irradiation of a cyprodinil aqueous solution combining LC/HRMS, LC/HRMS/MS and the use of a labeled compound. Their formations imply several photodegradation reactions, namely direct bond cleavages, cyclization, isomerization and hydroxylation. Most of the TPs exhibit a toxicity significantly higher than that of the parent molecule. Copyright © 2016 John Wiley & Sons, Ltd.

Structural elucidation of metolachlor photoproducts by liquid chromatography/high-resolution tandem mass spectrometry.

RATIONALE: Metolachlor is one of the most intensively used chloroacetanilide herbicides in agriculture. It has been detected in water; consequently, under UV-visible irradiation, it can be transformed in degradation products (TPs). The structures of TPs were elucidated by liquid chromatography/high-resolution tandem mass spectrometry (LC/HR-MS/MS). The potential toxicities of these TPs were estimated by in silico tests. METHODS: Aqueous solutions of metolachlor were irradiated in a self-made reactor equipped with a mercury vapor lamp. Analyses were carried out using high-performance liquid chromatography coupled to quadrupole time-of-flight (QTOF) mass spectrometer. High-resolution m/z measurements, MS/MS and isotopic labeling experiments allowed structural elucidation of metolachlor TPs. Their toxicities were estimated in silico, using the T.E.S.T. RESULTS: Ten major metolachlor photoproducts were characterized by LC/MS/MS after irradiation of metolachlor in aqueous solution. Elucidation of their chemical structures was identified using high-resolution measurements and MS/MS experiments. They resulted from the combination of dehalogenation, hydroxylation and cyclisation processes. The potential oral rat lethal dose (LD50) was assessed with QSAR tests for metolachlor and each photoproduct. Results indicate that most of the TPs are much more toxic than metolachlor. CONCLUSIONS: UV-vis irradiation of metolachlor in aqueous solution leads to the formation of ten photoproducts. QSAR estimations show that the location of added hydroxyl group(s) is of key relevance as regards to biological activity and that routine water analysis should take into account the TPs are more toxic than the parent molecule.

Isomerization of fenbuconazole under UV-visible irradiation - chemical and toxicological approaches.

RATIONALE: Fenbuconazole is a fungicide commonly used for the protection of vineyards, vegetables and grain crops. Under UV-visible irradiation, it undergoes isomerization through various cyclization processes. Isomeric structures were elucidated by liquid chromatography/high-resolution multiple-stage mass spectrometry (LC/HR-MS(n) ) coupling. The potential toxicities of these isomers were estimated by in silico tests. METHODS: Aqueous solutions of fenbuconazole and grapes treated with this fungicide were irradiated in a self-made reactor equipped with a mercury vapor lamp. Analyses were carried out using high-performance liquid chromatography (HPLC) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICRMS). High-resolution m/z measurements, multiple-stage mass spectrometry and isotopic labeling experiments allowed structural elucidation of the isomers of fenbuconazole. In silico toxicity estimations were carried out using the T.E.S.T. RESULTS: Seven isomers of fenbuconazole were detected after irradiation of the fungicide in aqueous solution; the major ones were also detected in the flesh of treated grapes irradiated under laboratory conditions. Elucidation of their chemical structures owing to high resolution measurements and multi-stage collision induced dissociation experiments allowed confirmation of photo-transformation pathways mainly dominated by cyclization processes. Photo-induced isomers exhibited higher potential toxicities than fenbuconazole for Daphnia magna and fathead minnow species. CONCLUSIONS: UV-visible irradiation of fenbuconazole in aqueous solution and on grapes leads to the formation of isomers, all of which being potentially much more toxic than the parent fungicide.

Combining gas phase electron capture and IRMPD action spectroscopy to probe the electronic structure of a metastable reduced organometallic complex containing a non-innocent ligand.

Combining electron capture dissociation mass spectrometry and infrared multiple photon dissociation action spectroscopy allows the formation, selection and characterisation of reduced metal complexes containing non-innocent ligands. Zinc complexes containing diazafluorenone ligands have been studied and the localisation of the single electron on the metal atom in the mono-ligated complex has been demonstrated.

Energy Resolved Mass Spectrometry for Interoperable Non-resonant Collisional Spectra in Metabolomics.

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 (ELab) 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 ELab 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 ELab 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 ELab 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.

Structure of sodiated polyglycines.

The intrinsic folding of peptides about a sodium ion has been investigated in detail by using infrared multiple photon dissociation (IRMPD) spectroscopy and a combination of theoretical methods. IRMPD spectroscopy was carried out on sodiated polyglycines G(n)-Na(+) (n=2-8), in both the fingerprint and N-H/O-H stretching regions. Interplay between experimental and computational approaches (classical and quantum) enables us to decipher most structural details. The most stable structures of the small peptides up to G(6)-Na(+) maximize metal-peptide interactions with all peptidic C=O groups bound to sodium. In addition, direct interactions between peptide termini are possible for G(6)-Na(+) and larger polyglycines. The increased flexibility of larger peptides leads to more complex folding and internal peptide structuration through γ or ß turns. A structural transition is found to occur between G(6)-Na(+) and G(7)-Na(+), leading to a structure with sodium coordination that becomes tri-dimensional for the latter. This transition was confirmed by H/D exchange experiments on G(n)-Na(+) (n=3-8). The most favorable hydrogen-bonding pattern in G(8)-Na(+) involves direct interactions between the peptide termini and opens the way to salt-bridge formation; however, there is only good agreement between experimental and computational data over the entire spectral range for the charge solvation isomer.

Structural influences on preferential oxazolone versus diketopiperazine b(2+) ion formation for histidine analogue-containing peptides.

Studies of peptide fragment ion structures are important to aid in the accurate kinetic modeling and prediction of peptide fragmentation pathways for a given sequence. Peptide b(2)(+) ion structures have been of recent interest. While previously studied b(2)(+) ions that contain only aliphatic or simple aromatic residues are oxazolone structures, the HA b(2)(+) ion consists of both oxazolone and diketopiperazine structures. The structures of a series of histidine-analogue-containing Xxx-Ala b(2)(+) ions were studied by using action infrared multiphoton dissociation (IRMPD) spectroscopy, fragment ion hydrogen-deuterium exchange (HDX), and density functional theory (DFT) calculations to systematically probe the influence of different side chain structural elements on the resulting b(2)(+) ion structures formed. The b(2)(+) ions studied include His-Ala (HA), methylated histidine analogues, including π-methyl-HA and τ-methyl-HA, pyridylalanine (pa) analogues, including 2-(pa)A, 3-(pa)A, and 4-(pa)A, and linear analogues, including diaminobutanoic acid-Ala (DabA) and Lys-Ala (KA). The location and accessibility of the histidine π-nitrogen, or an amino nitrogen on an aliphatic side chain, were seen to be essential for diketopiperazine formation in addition to the more typical oxazolone structure formation, while blocking or removal of the τ-nitrogen did not change the b(2)(+) ion structures formed. Linear histidine analogues, DabA and KA, formed only diketopiperazine structures, suggesting that a steric interaction in the HisAla case may interfere with the complete trans-cis isomerization of the first amide bond that is necessary for diketopiperazine formation.

Large-Scale Interlaboratory DI-FT-ICR MS Comparability Study Employing Various Systems.

Ultrahigh resolution mass spectrometry (UHR-MS) coupled with direct infusion (DI) electrospray ionization offers a fast solution for accurate untargeted profiling. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been shown to produce a wealth of insights into complex chemical systems because they enable unambiguous molecular formula assignment even if the vast majority of signals is of unknown identity. Interlaboratory comparisons are required to apply this type of instrumentation in quality control (for food industry or pharmaceuticals), large-scale environmental studies, or clinical diagnostics. Extended comparisons employing different FT-ICR MS instruments with qualitative direct infusion analysis are scarce since the majority of detected compounds cannot be quantified. The extent to which observations can be reproduced by different laboratories remains unknown. We set up a preliminary study which encompassed a set of 17 laboratories around the globe, diverse in instrumental characteristics and applications, to analyze the same sets of extracts from commercially available standard human blood plasma and Standard Reference Material (SRM) for blood plasma (SRM1950), which were delivered at different dilutions or spiked with different concentrations of pesticides. The aim of this study was to assess the extent to which the outputs of differently tuned FT-ICR mass spectrometers, with different technical specifications, are comparable for setting the frames of a future DI-FT-ICR MS ring trial. We concluded that a cluster of five laboratories, with diverse instrumental characteristics, showed comparable and representative performance across all experiments, setting a reference to be used in a future ring trial on blood plasma.

Sclareol and linalyl acetate are produced by glandular trichomes through the MEP pathway.

Sclareol, an antifungal specialized metabolite produced by clary sage, Salvia sclarea, is the starting plant natural molecule used for the hemisynthesis of the perfume ingredient ambroxide. Sclareol is mainly produced in clary sage flower calyces; however, the cellular localization of the sclareol biosynthesis remains unknown. To elucidate the site of sclareol biosynthesis, we analyzed its spatial distribution in the clary sage calyx epidermis using laser desorption/ionization mass spectrometry imaging (LDI-FTICR-MSI) and investigated the expression profile of sclareol biosynthesis genes in isolated glandular trichomes (GTs). We showed that sclareol specifically accumulates in GTs' gland cells in which sclareol biosynthesis genes are strongly expressed. We next isolated a glabrous beardless mutant and demonstrate that more than 90% of the sclareol is produced by the large capitate GTs. Feeding experiments, using 1-13C-glucose, and specific enzyme inhibitors further revealed that the methylerythritol-phosphate (MEP) biosynthetic pathway is the main source of isopentenyl diphosphate (IPP) precursor used for the biosynthesis of sclareol. Our findings demonstrate that sclareol is an MEP-derived diterpene produced by large capitate GTs in clary sage emphasing the role of GTs as biofactories dedicated to the production of specialized metabolites.

UV-visible photodegradation of naproxen in water - Structural elucidation of photoproducts and potential toxicity.

The UV-visible photodegradation of Naproxen (6-methoxy-α-methyl-2-naphthaleneacetic acid, CAS: 22204-53-1), one of the most used and detected non-steroidal anti-inflammatory drugs (NSAIDs) in the world, and its ecotoxicological consequences were investigated in an aqueous medium. The photo-transformation products were analyzed and the structures of photoproducts were elucidated using gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) and high-performance liquid chromatography coupled with ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS). Seven photoproducts were detected and characterized, photo-transformation mechanisms have been postulated to rationalize their formation under irradiation. In silico Q.S.A.R. (Quantitative Structure-Activity Relationship) toxicity predictions were performed with the Toxicity Estimation Software Tool (T.E.S.T.) and in vitro assays were carried out on Vibrio fischeri bacteria. Some of the obtained photoproducts exhibit higher potential toxicity than Naproxen itself but the whole toxicity of the irradiated solution is not of major concern.