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The secondary metabolome of Penicillium nordicum is poorly documented despite its frequent detection on contaminated food and its capacity to produce toxic metabolites such as ochratoxin A. To characterize metabolites produced by this fungi, we combined a full stable isotopes labeling with the dereplication of tandem mass spectrometry (MS/MS) data by molecular networking. First, the untargeted metabolomic analysis by high-resolution mass spectrometry of a double stable isotope labeling of P. nordicum enabled the specific detection of its metabolites and the unambiguous determination of their elemental composition. Analyses showed that infection of substrate by P. nordicum lead to the production of at least 92 metabolites and that 69 of them were completely unknown. Then, curated molecular networks of MS/MS data were generated with GNPS and MetGem, specifically on the features of interest, which allowed highlighting 13 fungisporin-related metabolites that had not previously been identified in this fungus and 8 that had never been observed in any fungus. The structures of the unknown compounds, namely, a native fungisporin and seven linear peptides, were characterized by tandem mass spectrometry experiments. The analysis of P. nordicum growing on its natural substrates, i.e. pork ham, turkey ham, and cheese, demonstrated that 10 of the known fungisporin-related metabolites and three of the new metabolites were also synthesized. Thus, the curation of data for molecular networking using a specific detection of metabolites of interest with stable isotopes labeling allowed the discovery of new metabolites produced by the food contaminant P. nordicum .

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.

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.

According to the World Health Organization, food safety is an essential public health priority. In this context, we report a relevant proof of feasibility for the indirect specific detection of bacteria in food samples using unlabeled phage amplification coupled to ESI mass spectrometry analysis and illustrated with the model phage systems T4 and SPP1. High-resolving power mass spectrometry analysis (including bottom-up and top-down protein analysis) was used for the discovery of specific markers of phage infection. Structural components of the viral particle and nonstructural proteins encoded by the phage genome were identified. Then, targeted detection of these markers was performed on a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. E. coli at 1 × 10(5), 5 × 10(5), and 1 × 10(6) CFU/mL concentrations was successfully detected after only a 2 h infection time by monitoring phage T4 structural markers in Luria-Bertani broth, orange juice, and French bean stew ("cassoulet") matrices. Reproducible detection of nonstructural markers was also demonstrated, particularly when a high titer of input phages was required to achieve successful amplification. This strategy provides a highly time-effective and sensitive assay for bacterial detection.

Protein domains involved in receptor heteromer formation are disordered and rich in the amino acids necessary for the formation of noncovalent complexes (NCX). We present mass spectral NCX data from proteins and protein receptors' epitopes obtained by combining ion mobility (IM) and MALDI. We focus on NCX involved in heteromer formation occurring between epitopes of the Dopamine D2 (D2R) and Adenosine A2A receptors (A2AR) as well as D2R and the α2 nicotinic (NR) receptor's subunit. The IM data yield information on the gas phase conformation of the singly charged NCX which are observed either directly from MALDI or as codesorbed neutrals that are subsequently postionized by a time-delayed excimer laser pulse directed onto a portion of the neutral plume created by the MALDI desorption laser. Imaging mass spectrometry of the matrix/epitope dried droplet surface shows that the acidic and basic epitopes and their NCX are found to be spatially collocated within regions as small as 25 × 50 μm(2). Subtle differences in the relative abundance of protonated and cationized NCX and epitopes are measured in spatial regions near the sodium-rich outer border of the droplet.

Metabolic profiles of biofluids obtained by atmospheric pressure ionization mass spectrometry-based technologies contain hundreds to thousands of features, most of them remaining unknown or at least not characterized in analytical systems. We report here on the annotation of the human adult urinary metabolome and metabolite identification from electrospray ionization mass spectrometry (ESI-MS)-based metabolomics data sets. Features of biological interest were first of all annotated using the ESI-MS database of the laboratory. They were also grouped, thanks to software tools, and annotated using public databases. Metabolite identification was achieved using two complementary approaches: (i) formal identification by matching chromatographic retention times, mass spectra, and also product ion spectra (if required) of metabolites to be characterized in biological data sets to those of reference compounds and (ii) putative identification from biological data thanks to MS/MS experiments for metabolites not available in our chemical library. By these means, 384 metabolites corresponding to 1484 annotated features (659 in negative ion mode and 825 in positive ion mode) were characterized in human urine samples. Of these metabolites, 192 and 66 were formally and putatively identified, respectively, and 54 are reported in human urine for the first time. These lists of features could be used by other laboratories to annotate their ESI-MS metabolomics data sets.

Epsilon toxin (ETX) is one of the most lethal toxins produced by Clostridium species and is considered as a potential bioterrorist weapon. Here, we present a rapid mass spectrometry-based method for ETX quantification in complex matrixes. As a prerequisite, naturally occurring prototoxin and toxin species were first structurally characterized by top-down and bottom-up experiments, to identify the most pertinent peptides for quantification. Following selective ETX immunoextraction and trypsin digestion, two proteotypic peptides shared by all the toxin forms were separated by ultraperformance liquid chromatography (UPLC) and monitored by ESI-MS (electrospray ionization-mass spectrometry) operating in the multiple reaction monitoring mode (MRM) with collision-induced dissociation. Thorough protocol optimization, i.e., a 15 min immunocapture, a 2 h enzymatic digestion, and an UPLC-MS/MS detection, allowed the whole quantification process including the calibration curve to be performed in less than 4 h, without compromising assay robustness and sensitivity. The assay sensitivity in milk and serum was estimated at 5 ng·mL(-1) for ETX, making this approach complementary to enzyme linked immunosorbent assay (ELISA) techniques.

Characterizing the conformation of biomolecules by mass spectrometry still represents a challenge. With their knotted structure involving a N-terminal macrolactam ring where the C-terminal tail of the peptide is threaded and sterically trapped, lasso peptides constitute an attractive model for developing methods for characterizing gas-phase conformation, through comparison with their unknotted topoisomers. Here, the kinetics of electron capture dissociation (ECD) of a lasso peptide, capistruin, was investigated by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry and compared to that of its branched-cyclic topoisomer, lactam-capistruin. Both peptides produced rather similar ECD spectra but showed different extent of H(•) transfer from c(i)' to z(j)(•) ions. Time-resolved double-resonance experiments under ECD conditions were performed to measure the formation rate constants of typical product ions. Such experiments showed that certain product ions, in particular those related to H(•) transfer, proceeded through long-lived complexes for capistruin, while fast dissociation processes predominated for lactam-capistruin. The formation rate constants of specific ECD product ions enabled a clear differentiation of the lasso and branched-cyclic topoisomers. These results indicate that the formation kinetics of ECD product ions constitute a new way to explore the conformation of biomolecules and distinguish between topoisomers and, more generally, conformers.

Apelin peptides were recently identified as endogenous ligands of the APJ receptor. It has been hypothesized that these peptides are initially provided to the newborn by nursing and might be involved in gastrointestinal tract development. As apelin peptides may have different effects on the APJ receptor as a function of their size, knowledge of their exact structure in early milk is essential to clarify their action in gastrointestinal tract development. Bovine colostrum is thought to contain high concentrations of a wide diversity of apelin peptides, but none of them have yet been rigorously characterized. To identify and monitor apelin peptides in bovine colostrum, we developed a cation exchange extraction step followed by untargeted liquid chromatography coupled to high resolution and high mass accuracy mass spectrometry (LTQ-Orbitrap). Using this approach, we characterized 46 endogenous apelin peptides in bovine colostrum, which varied in relative abundance from one colostrum to another. Mature as well as commercial milk samples were also studied. Taken together, our data demonstrate that the multiplicity and variability of apelin peptides are biologically relevant and change during milk maturation to reach a more constant composition in mature milk.

Recently, high-resolution mass spectrometry has been largely employed for compound identification, thanks to accurate mass measurements. As additional information, relative isotope abundance (RIA) is often needed to reduce the number of candidates prior to tandem MS(n). Here, we report on the evaluation of the LTQ-Orbitrap, in terms of accurate mass and RIA measurements for building further metabolomics spectral databases. Accurate mass measurements were achieved in the ppm range, using external calibration within 24 h, and remained at <5 ppm over a one-week period. The experimental relative abundances of (M+1) isotopic ions were evaluated in different data sets. First of all, 137 solutions of commercial compounds were analyzed by flow injection analysis in both the positive and negative ion modes. It was found that the ion abundance was the main factor impacting the accuracy of RIA measurements. It was possible to define some intensity thresholds above which errors were systematically <20% of their theoretical values. The same type of results were obtained with analyses from two biological media. Otherwise, no significant effect of ion transmission between the LTQ ion trap and the Orbitrap analyzer on RIA measurement errors was found, whereas the reliability of RIA measurements was dramatically improved by reducing the mass detection window. It was also observed that the signal integration method had a significant impact on RIA measurement errors, with the most-reliable results being obtained with peak height integrations. Finally, automatic integrations using the data preprocessing software XCMS and MZmine gave results similar to those obtained by manual integration, suggesting that it is relevant to use the RIA information in automatic elemental composition determination software from metabolomic peak tables.

The manganese(V)-nitrido polyoxometalate derivative [PW(11)O(39){Mn(V)N}](5-) has been synthesized by photochemical activation of the parent manganese(III)-azido derivative [PW(11)O(39)Mn(III)N(3)](5-). It was characterized by mass spectrometry, (31)P NMR, UV-vis, and IR spectroscopies. An electrochemistry study indicated that only the Mn(V) state was stable. The photoactivation of the manganese(III)-azido derivative proceeds through two competitive routes, yielding to the targeted product of photooxidation {Mn(V)N} or the undesirable product of photoreduction {Mn(II)L} (L = H(2)O, N(3)), depending on the photolysis conditions. A simplified photolysis mechanism involving two different excited states was proposed to account for the temperature and wavelength dependence.

Ricin, the toxin component of Ricinus communis is considered as a potential chemical weapon. Several complementary techniques are required to confirm its presence in environmental samples. Here, we report a method combining immunocapture and analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the accurate detection of different species of R. communis. Liquid environmental samples were applied to magnetic particles coated with a monoclonal antibody directed against the B-chain of the toxin. After acidic elution, tryptic peptides of the A- and B-chains were obtained by accelerated digestion with trypsin in the presence of acetonitrile. Of the 20 peptides observed by MALDI-TOF MS, three were chosen for detection ( m/ z 1013.6, m/ z 1310.6 and m/ z 1728.9, which correspond to peptides 161-LEQLAGNLR-169, 150-YTFAFGGNYDR-160, and 233-SAPDPSVITLENSWGR-248, respectively). Their selection was based on several parameters such as detection sensitivity, specificity toward ricin forms and absence of isotopic overlap with unrelated peptides. To increase assay reproducibility, stable isotope-labeled peptides were incorporated during the sample preparation phase. The final assay has a limit of detection estimated at approximately 50 ng/mL ( approximately 0.8 nM) of ricin in buffer. No interference was observed when the assay was applied to ricin-spiked milk samples. In addition, several varieties of R. communis or from different geographical origins were also shown to be detectable. The present assay provides a new tool with a total analytical time of approximately 5 h, which is particularly relevant in the context of a bioterrorist incident.

A strategy combining autocorrelation matrices and ultrahigh resolution mass spectrometry (MS) was developed to optimize the characterization of discriminating ions highlighted by metabolomics. As an example, urine samples from rats treated with phenobarbital (PB) were analyzed by ultrahigh-pressure chromatography with two different eluting conditions coupled to time-of-flight mass spectrometric detection in both the positive and negative electrospray ionization modes. Multivariate data analyses were performed to highlight discriminating variables from several thousand detected signals: a few hundred signals were found to be affected by PB, whereas a few tenths of them were linked to its metabolism. Autocorrelation matrices were then applied to eliminate adduct and fragment ions. Finally, the characterization of the ions of interest was performed with ultrahigh-resolution mass spectrometry and sequential MS(n) experiments, by using a LC-LTQ-Orbitrap system. The use of different eluting conditions was shown to drastically impact on the chromatographic retention and ionization of compounds, thus providing a way to obtain more exhaustive metabolic fingerprints, whereas autocorrelation matrices allowed one to focus the identification work on the most relevant ions. By using such an approach, 14 PB metabolites were characterized in rat urines, some of which have not been reported in the literature.

A nitroimidazole, molecularly imprinted polymer (MIP) was tested to extract four 5-nitroimidazoles (i.e., dimetridazole (DMZ), ipronidazole (IPZ), metronidazole (MNZ), and ronidazole (RNZ)) and three of their metabolites (i.e., DMZOH, IPZOH, and MNZOH) from egg powder samples. Various MIP templates were produced, and their selectivity was assessed on nitroimidazole standard solutions using liquid chromatography coupled with ultraviolet detection. The optimal cleanup was then used for the extraction of nitroimidazole in egg powder samples, and their quantification was achieved by isotope dilution LC-ESIMS/MS. The sample preparation entails a solubilization of the samples with water and acetonitrile followed by a MISPE cleanup step before LC-ESIMS/MS analysis. Data acquisition was achieved using selected reaction monitoring, and quantification was done with five deuterated analogues (i.e., DMZ- d(3), RNZ- d(3), IPZ- d(3), DMZOH- d(3), and IPZOH- d(3)). DMZOH- d(3) was used to quantify MNZ and MNZOH since they do not have their corresponding internal standards. The method was validated according to the European Union criteria by spiking experiments at concentration levels of 1, 2, and 3 microg/kg. At these three levels and for compounds having their own internal standards, acceptable performance data were obtained, with internal standard corrected recoveries ranging from 91 to 111%, and decision limits (CCalpha) and detection capabilities (CCbeta) were below 0.34 and 0.39 microg/kg, respectively.

We report the direct introduction of biological samples into a high-resolution mass spectrometer, the LTQ-Orbitrap, as a fast tool for metabolomic studies. A proof of concept study was performed on yeast cell extracts that were introduced into the mass spectrometer by using flow injection analysis, with an acquisition time of 3 min. Typical mass spectra contained a few thousand m/z signals, 400 of which were found to be analytically relevant (i.e., their intensity was 3-fold higher than that of the background noise and they occurred in at least 60% of the acquisition profiles under identical experimental conditions). The method was validated by studies of the matrix effect, linearity, and intra-assay precision. Accurate mass measurements in the Orbitrap discriminated between isobaric ions and also indicated the elemental composition of the ions of interest with mass errors below 5 ppm, for identification purposes. The proposed structures were then assessed by MSn experiments via the linear ion trap, together with accurate mass determination of the product ions in the Orbitrap analyzer. When applied to the study of cadmium toxicity, the method was as effective as that initially developed by using LC/ESI-MS/MS for a targeted approach. The same metabolic fingerprints were also subjected to multivariate statistical analyses. The results highlighted a reorganization of amino acid metabolism under cadmium conditions in order to increase the biosynthesis of glutathione.

In this study, we show that liquid chromatography coupled with tandem mass spectrometry provides a sensitive, specific, and accurate absolute quantification of Erbitux, a human:murine chimeric mAb used for the treatment of colorectal cancer. Micrometric magnetized beads, functionalized with soluble epidermal growth factor receptor (sEGFR), the pharmacological target of Erbitux, were used for specific immunocapture of Erbitux allowing assessment of the antibody's biological potency and sample purification. Following digestion with trypsin, specific peptides from light and heavy chains were monitored in the selected reaction monitoring (SRM) mode. Assay variability below 20% was provided through optimization of the digestion step and rigorous monitoring of the whole analytical process using an appropriate internal standard. The 20 ng/mL lower limit of quantification was similar to that of ELISA methods. These results show that this mass spectrometric approach is a potential alternative for pharmacokinetic evaluation of mAbs during clinical development.

A simple and fast selective extraction of the antibiotic chloramphenicol (CAP) from milk (raw milk, skimmed milk, and milk powder) using a molecularly imprinted polymer (MIP) sorbent is described. The method entails a single centrifugation step prior to loading the supernatant onto the MIP cartridge and subsequent elution with a mixture of solvents. CAP was further analyzed by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) operating in negative ionization acquisition mode. The advantages of the MIP approach were assessed by comparing the data generated from a classical solid-phase and liquid-liquid extractions procedure, previously developed in our laboratory. A better recovery of CAP due to an enhanced selectivity and a faster turnaround time (18 samples processed within 3 h compared to 8 h with the classical approach) were evidenced when using the MIP cleanup. The analysis of CAP in raw milk was further validated according to the 2002/657/EC European Union criteria for the analysis of veterinary drug residues at the minimum required performance limit (MRPL) of 0.3 microg/kg, using CAP-d(5) as internal standard. Non-internal-standard corrected recovery values ranged between 50% and 87% over the range of concentrations considered. The decision limit (CCalpha) and detection capability (CCbeta) were calculated to be 0.06 and 0.10 microg/kg, respectively.

Formalin fixation, generally followed by paraffin embedding, is the standard and well-established processing method employed by pathologist. This treatment conserves and stabilizes biopsy samples for years. Analysis of FFPE tissues from biopsy libraries has been, so far, a challenge for proteomics biomarker studies. Herein, we present two methods for the direct analysis of formalin-fixed, paraffin-embedded (FFPE) tissues by MALDI-MS. The first is based on the use of a reactive matrix, 2,4-dinitrophenylhydrazine, useful for FFPE tissues stored less than 1 year. The second approach is applicable for all FFPE tissues regardless of conservation time. The strategy is based on in situ enzymatic digestion of the tissue section after paraffin removal. In situ digestion can be performed on a specific area of the tissue as well as on a very small area (microdigestion). Combining automated microdigestion of a predefined tissue array with either in situ extraction prior to classical nanoLC/MS-MS analysis or automated microspotting of MALDI matrix according to the same array allows the identification of both proteins by nanoLC-nanoESI and MALDI imaging. When adjacent tissue sections are used, it is, thus, possible to correlate protein identification and molecular imaging. These combined approaches, along with FFPE tissue analysis provide access to massive amounts of archived samples in the clinical pathology setting.

The toxin ricin is a biological weapon that may be used for bioterrorist purposes. As a member of the group of ribosome-inactivating proteins (RIPs), ricin has an A-chain possessing N-glycosidase activity which irreversibly inhibits protein synthesis. In this paper, we demonstrate that provided appropriate sample preparation is used, this enzymatic activity can be exploited for functional ricin detection with sensitivity similar to the best ELISA and specificity allowing application to environmental samples. Ricin is first captured by a monoclonal antibody directed against the B chain and immobilized on magnetic beads. Detection is then realized by determination of the adenine released by the A chain from an RNA template using liquid chromatography coupled to tandem mass spectrometry. The immunoaffinity step combined with the enzymatic activity detection leads to a specific assay for the entire functional ricin with a lower limit of detection of 0.1 ng/mL (1.56 pM) after concentration of the toxin from a 500 microL sample size. The variability of the assay was 10%. Finally, the method was applied successfully to milk and tap or bottled water samples.

Direct tissue analysis using MALDI-MS allows the generation of profiles while maintaining the integrity of the tissue, displaying cellular localizations and avoiding tedious extraction and purification steps. However, lower spectral quality can result from direct tissue analysis due to variations in section thickness, the nature of the tissue, and the limited access to peptides/proteins due to high lipid content. To improve signal sensitivity, we have developed a tissue-washing procedure using organic solvents traditionally used for lipid extraction, i.e., CHCl3, hexane, toluene, acetone, and xylene. The increased detection for peptides/proteins (m/z 5000-30,000) is close to 40% with chloroform or xylene, and 25% with hexane, while also improving sample reproducibility for each solvent used in the present study. This strategy improved matrix cocrystallization with tissue peptides/proteins and more importantly with cytoplasmic proteins without delocalization. The extracted lipids were characterized by nanoESI-QqTOF/MS/MS using the precursor ion mode, lithium adducts, or both and were identified as phospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and lysophosphatidylinositol, confirming membrane lipid extraction from the tissues.

LC/ESI-MS/MS is a promising alternative to immunoassays in improving the analysis of recombinant therapeutic proteins in biological fluids for toxicity and pharmacokinetics purposes. To assess the sensitivity and validation issues associated with this technique, we use here as a model EPI-hNE4, a 56-amino acid recombinant protein, and demonstrate that a method based on tandem mass spectrometry combined with liquid chromatography and electrospray interface can reach sensitivity similar to that of ELISA but without its potential cross-reactivity. For this purpose, a triple quadrupole mass spectrometer operating in positive ion and single reaction monitoring mode with transition, m/z 1040 --> 1224.5, was used for selective peak detection. Particular issues related to the internal standard, i.e., elution and ionization patterns similar to the protein without stable isotope labeling, and to analytical interference due to endogenous binding antibodies were addressed. A limit of quantification in human or monkey plasma of 5 ng/mL was reached with a sample volume of 100 microL, corresponding to 40 fmol injected into the HPLC column. Intra- and interassay precision and accuracy were below 15%. No matrix effect was detected.

The incorporation of lanthanides into polyoxometalates provides entry to new classes of potentially useful materials that combine the intrinsic properties of both constituents. To utilize the [alpha1-Ln(H2O)4P2W17O61]7- species in applications of catalysis and development of luminescent materials, the chemistry of this family of lanthanide polyoxometalates in organic solvents has been developed. Organic-soluble polyoxometalate-lanthanide complexes TBA5H2[alpha1-Ln(H2O)4P2W17O61] (Ln = La(III), Sm(III), Eu(III), Yb(III)) were prepared and characterized by elemental analysis, acid-base titration, IR, 31P NMR, and mass spectrometry. The synthetic procedure involves a cation metathesis reaction in aqueous solution under strict pH control. A solid-liquid-phase transfer protocol yielded a unique species (TBA)8K3[Yb(alpha1-YbP2W17O61)2] with three ytterbium ions and two [alpha1-P2W17O61]10- polyoxotungstates. A centrosymmetric dimeric complex [{alpha1-La(H2O)4P2W17O61}2]14- was crystallized from aqueous solution and characterized by X-ray diffraction. ESI mass spectral analysis of the complexes TBA5H2[alpha1-Ln(H2O)4P2W17O61] shows that similar dimers exist in organic solution, in particular for the early lanthanides. Fragmentation in the mass spectrometer of the complexes from dry acetonitrile solution involves double protonation of an oxo ligand and loss of one water molecule. Low mass tungstate fragments combine into [(WO3)n]2- (n = 1-5) ions and their condensation products with phosphate. Reaction of TBA5H2[alpha1-Eu(H2O)4P2W17O61] with 1,10-phenanthroline or 2,2'-bipyridine showed an increase of the europium luminescence. This result is explained by the formation of a ternary complex of [alpha1-Eu(H2O)4P2W17O61]7- and two sensitizing ligands.

Direct analysis of tissue by MALDI-MS allows the acquisition of its biomolecular profile while maintaining the integrity of the tissue, giving cellular localization, and avoiding tedious extraction and purification steps. However, direct tissue analysis generally leads to some extent to a lowered spectral quality due to variation in thickness, freezing tissue date, and nature of the tissue. We present here new technical developments for the direct tissue analysis of peptides with ionic liquid made of matrix mixtures (alpha-cyano-4-hydroxycinnamic acid (CHCA)/2-amino-4-methyl-5-nitropyridine and alpha-cyano-4-hydroxycinnamic acid/N,N-dimethylaniline (CHCA/DANI)). The properties of these direct tissue analysis matrixes, especially CHCA/aniline when compared to CHCA, 2,5-dihydroxybenzoic acid, and sinapinic acid, are as follows: (1) better spectral quality in terms of resolution, sensitivity, intensity, noise, number of compounds detected, and contaminant tolerance, (2) better crystallization on tissues, i.e., coverage capacity, homogeneity of crystallization, homogeneity of crystal sizes, and time of crystallization, (3) better analysis duration in term of vacuum stability, (4) better resistance to laser irradiation especially for high-frequency lasers, (5) better ionic yield in negative mode, and (6) enough fragmentation yield to use the PSD mode on sections to get structural information. Applied to MALDI imaging on a MALDI LIFT-TOF with a 50-Hz laser frequency, these ionic matrixes have allowed the realization of a new type of image in both polarities and reflector mode using the same tissue section. These results give a new outlook on peptide tissue profiling by MS, characterization of compounds from tissue slices, and MALDI-MS high-quality imaging.

Hemoglobin-based oxygen carriers (HBOCs) are being developed for the medical field, but because they could increase an athlete's performance, they are misapplied for doping purposes. We previously presented a screening method to detect Oxyglobin (Biopure Corp.) and PolyHeme (Northfield Laboratories Inc.) in serum samples using total acid hydrolysis followed by electrospray mass spectrometry analyses. An alternative mass spectrometric method involving enzymatic hydrolysis is here presented. Digestion of Oxyglobin by endoproteinase Glu-C and LC/MS analyses of the mixture allowed the detection of unique peptidic fragments in comparison with a bovine hemoglobin digest. Tandem mass spectrometry experiments of these peptide ions were performed, and two specific species were actually identified as the N-terminal enzymatic fragment of the beta chain carrying two different modifications. Sequential MS3 experiments using an ion trap mass spectrometer permitted us to locate the chemical modification by the glutaraldehyde on the NH2-terminal group and to propose a structure for the modified peptides. In another set of experiments, screening of these two diagnostic ions into Oxyglobin-spiked serums using precursor ion scan mode in a triple quadrupole instrument allowed the detection of this HBOC with a detection limit of 2 g L(-1).

Metabolomics, i.e., the global analysis of cellular metabolites, is becoming a powerful tool for gaining insights into biological functions in the postgenomic context. However, absolute quantitation of endogenous metabolites in biological media remains an issue, and available technologies for the analysis of metabolome still lack robustness and accuracy. We describe here a new method based on liquid chromatography-mass spectrometry and (15)N uniform metabolic labeling of Saccharomyces cerevisiae for accurate and absolute quantitation of nitrogen-containing cell metabolites in metabolic profiling experiments. As a proof of concept study, eight sulfur metabolites involved in the glutathione biosynthesis pathway (i.e., cysteine, homocysteine, methionine, gamma-glutamylcysteine, cystathionine, reduced and oxidized forms of glutathione, and S-adenosylhomocysteine) were simultaneously quantified. The analytical method has been validated by studies of stability, selectivity, precision, and linearity and by the determination of the limits of detection and quantification. It was then applied to the analysis of extracts from cadmium-treated yeasts. In these conditions, the intracellular concentrations of most of the metabolites involved in the glutathione biosynthesis pathway were increased when compared to control extracts. These data correlate with previous proteomic results and also underline the importance of glutathione in cadmium detoxication.

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).

Reaction of [Bu(4)N](4)[H(3)PW(11)O(39)] with [Re(NPh)Cl(3)(PPh(3))(2)], in acetonitrile and in the presence of NEt(3), provided the first Keggin-type organoimido derivative [Bu(4)N](4)[PW(11)O(39)(ReNPh)] (Ph = C(6)H(5)) (1). The functionalization was clearly demonstrated by various techniques including (1)H and (14)N NMR, electrochemistry, and ESI mass spectrometry. Conditions for the formation of 1 are also discussed.

The structure of a two-chain peptide formed by the treatment of the potent antimicrobial peptide microcin J25 (MccJ25) with thermolysin has been characterized by NMR spectroscopy and mass spectrometry. The native peptide is 21 amino acids in size and has the remarkable structural feature of a ring formed by linkage of the side chain of Glu8 to the N-terminus that is threaded by the C-terminal tail of the peptide. Thermolysin cleaves the peptide at the Phe10-Val11 amide bond, but the threading of the C-terminus through the N-terminal ring is so tight that the resultant two chains remain associated both in the solution and in the gas phases. The three-dimensional structure of the thermolysin-cleaved peptide derived using NMR spectroscopy and simulated annealing calculations has a well-defined core that comprises the N-terminal ring and the threading C-terminal tail. In contrast to the well-defined core, the newly formed termini at residues Phe10 and Val11 are disordered in solution. The C-terminal tail is associated to the ring both by hydrogen bonds stabilizing a short beta-sheet and by hydrophobic interactions. Moreover, unthreading of the tail through the ring is prevented by the bulky side chains of Phe19 and Tyr20, which flank the octapeptide ring. This noncovalent two-peptide complex that has a remarkable stability in solution and in highly denaturing conditions and that survives in the gas phase is the first example of such a two-chain peptide lacking disulfide or interchain covalent bonds.

The lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is generated as a consequence of oxidative stress and can readily react with nucleophilic sites of proteins (e.g., histidine residues), mainly via a Michael addition. The formation of such lipid-protein conjugates can alter protein properties and biological functions, thus leading to highly deleterious effects. The present work describes a rapid (very limited sample preparation) and sensitive (low-femtomole range) procedure to identify HNE-modified peptides (Michael adducts) within unfractionated tryptic digests. The protocol involves the formation of dinitrophenylhydrazones of the Michael adducts, when using 2,4-dinitrophenylhydrazine as reactive matrix, followed by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives present high desorption/ionization yield and can thus be preferentially detected compared to unmodified peptides. The MALDI mass spectrum obtained is therefore drastically different from the one obtained with the classical 4-hydroxy-alpha-cyanocinnamic acid matrix. Moreover, the presence of HNE, or more generally speaking carbonylated peptides, could be highlighted by 180 mass units differences (corresponding to the dinitrophenylhydrazone moiety) between these two MALDI mass spectra. Further information (e.g., localization/identification of the modified residues, peptide sequences) could be obtained by performing MALDI postsource decay (or electrospray) MS/MS experiments on the ions of interest.

[reaction: see text]. The Magnus Pauson-Khand (PK) mechanism has been studied for the first time by electrospray ionization coupled to tandem mass spectrometry. It has been found that loss of CO from the PK complex precedes olefin coordination and insertion.

Characterization of fluorinated polymers in MALDI is often unsuccessful because commonly used matrixes, such as 2,5-dihydroxybenzoic acid, Indole acrylic acid, alpha-cyano-4-hydroxycinnamic acid, etc., do not desorb/ionize fluorinated polymers efficiently. This could be in part attributed to the unfavorable interaction between the matrix molecules and fluorinated oligomers due to differences in their hydrophobicities. Moreover, the relative cation affinity between the matrix molecules and the fluorinated oligomers may not favor the gas-phase cationization process of the fluorinated oligomers. To overcome these limitations, fluorinated derivates of benzoic acid (pentafluorobenzoic acid) and cinnamic acid (Pentafluoro cinnamic acid) were employed for the desorption/ionization of perfluoropolyethers. Presence of fluorine atoms in the matrix might improve the interaction between the matrix and perfluoroether during the crystallization or ionization step. With a pentafluorobenzoic acid matrix, intact silver cationized oligomers were desorbed, whereas with a pentafluorocinnamic acid matrix, loss of end group was observed. This loss could be rationalized by the dissociation of the silver cationized oligomers via an ion-dipole mechanism. This work shows the possibility of characterizing yet another important class of fluorinated polymer by MALDI-TOFMS.

Two methods based on specific immunoaffinity enrichment followed by electrospray ionization (ESI) mass spectrometry (MS) have been developed for the specific analysis of 4-hydroxy-2-nonenal (HNE)- and malondialdehyde (MDA)-modified proteins (Michael and Schiff base adducts, respectively). Anti-HNE antibodies were immobilized on CNBr-activated sepharose, and the immunosorbent produced was used for the enrichment of HNE-adducted peptides originating from a model peptide modification and a tryptic digest of modified apomyoglobin. A further immunosorbent was produced by anti-dinitrophenyl immobilization and assayed for selective extraction of peptides modified with HNE and MDA that were initially converted to their respective hydrazones. Subsequent analysis and characterization of the different purified fractions by ESI-MS (MS/MS) revealed that the two immunosorbents enable efficient and specific enrichment of the carbonyl adducted proteins. This approach lowers substantially the detection limit of such modifications and, thus, enables better assessment and characterization of carbonyl modifications in biological and food systems.

Pyrolysis coupled to metastable atom bombardment (MAB) and time-of-flight mass spectrometry (TOFMS) is used for generating mass spectra from bovine urine samples obtained from cattle treated with anabolic steroids. These spectra constitute fingerprints, which can be discriminated by multivariate statistical analysis. Four main conclusions can be drawn from this work: (i) The use of different metastable gases, such as Xe*, Kr*, or N2*, as an energy-tunable ionization beamline allows control of the internal energy and the dissociation processes of the produced odd electron molecular ions, thus giving rise to complementary mass spectra fingerprints. (ii) A variable transformation depending on the biofluid matrix suitably contracts the frequency distribution of the generated data for low m/z ratios holding information related to endogenous metabolites encountered in urine. (iii) Coupling variable selection to statistical pattern recognition methods results in low error rates (< 1%) for predicting MAB mass fingerprints, especially using lineardiscriminant analysis (LDA). (iv) LDA discriminates controls from treated animals and also correlates to quantitative physiological responses induced by anabolic steroids. This work shows that Py-MAB-TOFMS could be a suitable method for complementary monitoring anabolic use in sports, medicine, and cattle breeding, as well as monitoring many other long-lasting although weak physiological disruptions.

Although fluorinated polymers are widely used in different applications, they are rarely investigated by "soft ionization" techniques such as matrix-assisted laser desorption/ionization and electrospray ionization (ESI). We report here, the desorption and fragmentation of poly(vinylidene) fluoride (PVDF) telomers in an ion trap mass spectrometer coupled to an ESI source. Protonated and lithiated telomers under collision-induced dissociation resonant excitation show mainly HF losses. Fragmentation of protonated telomers can be rationalized by a double proton-transfer mechanism and that of lithiated and anionic telomers by an ion-dipole mechanism. Both mechanisms predict the formation of a stable aromatic or a linear conjugated species, respectively. For lithiated telomers, we could determine the degree of polymerization (n) from the product ion abundance in MS2 experiments. The nature of the end group plays a substantial role in orienting the fragmentation of the PVDF ions. It is interesting to note that, in MS2 experiments, Li+ and F- act as catalysts in the fragmentation of PVDF telomers. Fragmentation of the PVDF telomer backbone was not observed under any experimental conditions.

Estrogen 2,3- and 3,4-quinones are reactive species toward nucleophiles and Michael acceptors. As such, they can bind to DNA and induce cellular damages. As an alkylation model, reactions of estradiol-2,3-quinone with deoxyribonucleosides were previously studied by mass spectrometry. In this work, estrogen-deoxyribonucleoside adducts were synthesized by reaction of 17beta-estradiol-2,3-quinone with deoxyguanosine or deoxyadenosine and analyzed by NMR and LC-MS(n)() in order to determine the structure and the stereochemistry of the resulting covalent adducts. Although estradiol- and estrone-2,3-quinones were previously thought to give mainly stable adducts, identification of depurinating adducts with both nucleosides, i.e., 2-OHE(2)-6(alpha,beta)-N7Gua and 2-OHE(2)-6(alpha,beta)-N7Ade, was unambiguously obtained. This is of particular interest since depurinating adducts are generated from DNA, and therefore, their amount should be correlated to the parallel formation of apurinic sites, which might play an important role in the cancer initiation process. Besides, a byproduct, i.e., 2-hydroxy-11-oxo-estradiol, corresponding to an unstable alkylation product of 2-hydroxyestradiol has been unambiguously identified and is indicative of a plausible addition process at the C9 position of catechol estrogens. The synthetic adducts will be useful as reference compounds to further elucidate the structure of adducts formed by reaction of estrogen metabolites with DNA or oligonucleotides.

Negative ion chemical ionization was carried out using a quadrupole ion trap mass spectrometer with selected reactant negative ions, primarily injected from a homemade dual EI/CI external ion source. Hence, selective ion/molecule reactions were provided according to the reaction time, which induce a greater control over bimolecular ionization mechanisms than in conventional a high-pressure ion source combined with beam instruments, where several competitive ionization processes take place mainly due to source conditions (e.g., temperature, pressure, and repeller). By selecting the reactant ions, ion/molecule reactions were specifically produced (i.e., charge exchange, proton transfer, nucleophilic substitution, and/or alpha-beta elimination) with several organic target compounds. Gas-phase reactivity of phosphorus- and nitrogen-containing compounds (such as phosphonates as representative for chemical warfare agents and phosphorothionates, phosphorodithionates, and triazines for pesticides) as well as dinitro aromatic compounds (for pesticides) has been explored, in the present work, to ensure further unambiguous detection.

Characterization of synthetic polymers by Matrix assisted laser desorption (MALDI) is limited by the solubility of different oligomers in a suitable solvent, and the fingerprint of the mass spectrum is affected by the properties of solvents employed (eg., pH, secondary solvents, evaporation) during sample target preparation. If solvents are not used during sample target preparation, then solvent properties should not play an important role in determining the quality of the MALDI mass spectrum. We report here two solventless approaches for sample target preparation. It was observed that Poly(ethylene glycol) 6000 (PEG) showed the same molecular mass distribution in different modes of sample target preparation. Fluorinated polymer used in these studies was affected by sample target preparation protocol and by target surface. Pyrolysis of PEG oligomers was observed in all the methods of sample target preparation. The desorbed high mass neutral oligomers fragment to give small oligomers which are then cationized by the desolvation of the cationized matrix clusters. Moreover, the origin of the matrix clusters (i.e., formed in the condensed phase or in the gas phase) determines the relative intensities of PEG oligomers cationized by sodium or potassium.

A new combination of a dual EI/CI ion source with a quadrupole ion trap mass spectrometer has been realized in order to efficiently produce negative ions in the reaction cell. Analysis of volatile compounds was performed under negative ion chemical ionization (NICI) during a reaction period where selected reactant negative ions, previously produced in the external ion source, were allowed to interact with molecules, introduced by hyphenated techniques such as gas chromatography. The O2*-, CH3O-, and Cl- reactant ions were used in this study to ensure specific ion/molecule interactions such as proton transfer, nucleophilic displacement, or charge exchange processes, respectively leading to even-electron species, i.e., deprotonated [M - H]- molecules, diagnostic [M - R]- ions, or odd-electron M*- molecular species. The reaction orientation depends on the thermochemistry of reactions within kinetic controls. First analytical results are presented here for the trace-level detection of several contaminants under NICI/Cl- conditions. Phosphorus-containing compounds (malathion, ethyl parathion, and methyl parathion as representative for pesticides) and nitro-containing compounds (2,4,6-trinitrotoluene for explosive material) have been chosen in order to explore the analytical ability of this promising instrumental coupling.

An analytical method for the determination of glyphosate and its principal metabolite, aminomethylphosphonic acid (AMPA), in water of different hardnesses (5, 20, and 30 degrees DH, french hardness) has been developed. Samples were fortified at different levels (0.05, 0.1, 1, and 5 microg/L) and were purified by column chromatography on ion-exchange resins. After derivatization with TFAA/HFB mixture, the derivatives were quantified by using capillary gas chromatography with an ion-trap tandem mass spectrometric detector. Analytical conditions for MS/MS detection were optimized, and the quantification was carried out on the sum of areas of the three most representative ions: m/z 283, 223, and 181 for AMPA and m/z 440, 321, and 261 for glyphosate. The limit of quantification was demonstrated to be at 0.05 microg/L for each compound. The mean recovery value and the relative standard deviation (n = 65) were 93 and 12% for AMPA and 95 and 13% for glyphosate.

Electrospray ionization mass spectrometry was tested for its potential use in the quantification of monobutyl phosphate (H2MBP) and dibutyl phosphate (HDBP), two degradation products of tributyl phosphate (TBP), the extractant used in the nuclear fuel reprocessing known as the PUREX process. Detection and quantification of these phosphate esters by electrospray in positive and negative ionization mode are reported in this study. This fast and reliable method, which does not require any preliminary sample extraction, appears to be very attractive for process control. Negative ionization mode gave abundant [M - H]- ions for both HDBP and H2MBP products. Thus, the concentration of H2MBP between 0.1 and 10 g/L in concentrated aqueous nitrate solutions can be precisely determined. Moreover, the concentration of HDBP up to 1 g/L in a TBP matrix was evaluated in this mode. For HDBP concentrations below 1 g/L, detection in the positive ionization mode appeared to be attractive. TBP and HDBP cluster detection allowed quantitative HDBP determination. Indeed, small amounts of HDBP in commercial TBP (60 mg/L) could be directly quantified using the specific [2TBP, HDBP + H]+ cluster at m/z 743.

A protocol including 2D SDS-PAGE, electroblotting proteins onto nitrocellulose membranes, and CNBr cleavage, followed by MALDI-MS analysis of intact proteins and peptide fragments and a database search, has been optimized and applied to the rapid identification of the Escherichia coli response to hypochlorous acid. The methodology has proved to be efficient from the point of view of sensitivity (picomole range) and selectivity. In particular, MALDI analysis of proteins and CNBr fragments by directly dissolving the membrane in an acetone solution of matrix, without previous elution, is reliable and reproducible. The accuracy of the MW determination is somewhat reduced compared to that of methods involving elution and purification of proteins and digests; nevertheless, the utilization of large MW windows combined with the pI entry in database searches had allowed, for most of the spots, the selection of only one protein candidate. Finally, 19 proteins exhibiting a response to hypochlorous acid stress have been confirmed or identified on the basis of this protocol.

Surface-induced dissociation (SID) has been used to produce daughter ion spectra of small protonated peptides generated by fast atom bombardment (FAB). The relative abundances of daughter ions depends critically upon the energy of the ion/surface collision. A wide array of decomposition processes may be observed using ELAB collision energies in the range 10-20 eV. At approximately 13-eV collision energy, the variety of decomposition processes is maximized for the small peptides studied; hence, maximum structural information may be deduced. Collisionally-activated dissociations (CAD) using argon gas and the identical protonated peptides could not produce as large an array of daughter ions in a constant condition experiment. An apparent contradiction is thereby posed because SID is known to produce a narrow distribution of ion internal energies relative to CAD. This apparent contradiction is resolved by considering the rather large kinetic energy spread of ions leaving the FAB source. For the SID process, this large initial kinetic energy distribution is converted into a significantly wider spread in center-of-mass collision energy, leading to a broader variety of decomposition processes (high and low energy) compared to CAD.

The stereochemical course of the vitamin K dependent carboxylation has been elucidated using a (4S)-4-fluoroglutamyl-containing pentapeptide as a substrate. The absolute configuration of the [13C]-4-carboxy-4-fluoroglutamate obtained when the carboxylation was carried out with 13C-labeled sodium bicarbonate, was determined after reduction of the [13C]-4-carboxy-4-fluoroglutamyl residue into 4-fluoro-5,5'-dihydroxyleucine, hydrolysis, lactonization, and peracetylation. The absolute configuration at C-4 was determined to be S by locating the 13C label in the lactone ring of the trans isomeric lactone and in the hydroxymethyl group of the cis isomer following HPLC separation of both isomers and analysis by GC/MS/MS techniques. It follows that the vitamin K dependent carboxylation occurs with inversion of configuration.