High-resolution mass spectrometry applied to the study of metabolome modifications in various chicken tissues after amoxicillin administration

The performance of high resolution accurate mass spectrometry (HRMS) operating in full scan MS mode was investigated for the quantitative determination of amoxicillin (AMX) as well as qualitative analysis of metabolomic profiles in tissues of medicated chickens. The metabolomic approach was exploited to compile analytical information on changes in the metabolome of muscle, kidney and liver from chickens subjected to a pharmacological program with AMX. Data consisting of m/z features taken throughout the entire chromatogram were extracted and filtered to be treated by Principal Component Analysis. As a result, it was found that medicated and non-treated animals were clearly clustered in distinct groups. Besides, the multivariate analysis revealed some relevant mass features contributing to this separation. In this context, recognizing those potential markers of each chicken class was a priority research for both metabolite identification and, obviously, evaluation of food quality and health effects associated to food consumption.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial strains.

Conventional methods are sometimes insufficient to identify human bacterial pathogens, and alternative techniques, often molecular, are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species having required 16S rRNA gene sequencing. MALDI-TOF MS might represent an alternative to 16S rRNA gene sequencing.

Quantify this! Report on a round table discussion on quantitative mass spectrometry in proteomics.

Following the success of the first round table in 2001, the Swiss Proteomic Society has organized two additional specific events during its last two meetings: a proteomic application exercise in 2002 and a round table in 2003. Such events have as their main objective to bring together, around a challenging topic in mass spectrometry, two groups of specialists, those who develop and commercialize mass spectrometry equipment and software, and expert MS users for peptidomics and proteomics studies. The first round table (Geneva, 2001) entitled "Challenges in Mass Spectrometry" was supported by brief oral presentations that stressed critical questions in the field of MS development or applications (Stöcklin and Binz, Proteomics 2002, 2, 825-827). Topics such as (i) direct analysis of complex biological samples, (ii) status and perspectives for MS investigations of noncovalent peptide-ligant interactions; (iii) is it more appropriate to have complementary instruments rather than a universal equipment, (iv) standardization and improvement of the MS signals for protein identification, (v) what would be the new generation of equipment and finally (vi) how to keep hardware and software adapted to MS up-to-date and accessible to all. For the SPS'02 meeting (Lausanne, 2002), a full session alternative event "Proteomic Application Exercise" was proposed. Two different samples were prepared and sent to the different participants: 100 micro g of snake venom (a complex mixture of peptides and proteins) and 10-20 micro g of almost pure recombinant polypeptide derived from the shrimp Penaeus vannamei carrying an heterogeneous post-translational modification (PTM). Among the 15 participants that received the samples blind, eight returned results and most of them were asked to present their results emphasizing the strategy, the manpower and the instrumentation used during the congress (Binz et. al., Proteomics 2003, 3, 1562-1566). It appeared that for the snake venom extract, the quality of the results was not particularly dependant on the strategy used, as all approaches allowed Lication of identification of a certain number of protein families. The genus of the snake was identified in most cases, but the species was ambiguous. Surprisingly, the precise identification of the recombinant almost pure polypeptides appeared to be much more complicated than expected as only one group reported the full sequence. Finally the SPS'03 meeting reported here included a round table on the difficult and challenging task of "Quantification by Mass Spectrometry", a discussion sustained by four selected oral presentations on the use of stable isotopes, electrospray ionization versus matrix-assisted laser desorption/ionization approaches to quantify peptides and proteins in biological fluids, the handling of differential two-dimensional liquid chromatography tandem mass spectrometry data resulting from high throughput experiments, and the quantitative analysis of PTMs. During these three events at the SPS meetings, the impressive quality and quantity of exchanges between the developers and providers of mass spectrometry equipment and software, expert users and the audience, were a key element for the success of these fruitful events and will have definitively paved the way for future round tables and challenging exercises at SPS meetings.

Contribution of isotope ratio mass spectrometry to the investigation of improvised explosives: isotopic study of black powders ans ammonium nitrates

The establishment of legislative rules about explosives in the eighties has reduced the illicit use of military and civilian explosives. However, bomb-makers have rapidly taken advantage of substances easily accessible and intended for licit uses to produce their own explosives. This change in strategy has given rise to an increase of improvised explosive charges, which is moreover assisted by the ease of implementation of the recipes, widely available through open sources. While the nature of the explosive charges has evolved, instrumental methods currently used in routine, although more sensitive than before, have a limited power of discrimination and allow mostly the determination of the chemical nature of the substance. Isotope ratio mass spectrometry (IRMS) has been applied to a wide range of forensic materials. Conclusions drawn from the majority of the studies stress its high power of discrimination. Preliminary studies conducted so far on the isotopic analysis of intact explosives (pre-blast) have shown that samples with the same chemical composition and coming from different sources could be differentiated. The measurement of stable isotope ratios appears therefore as a new and remarkable analytical tool for the discrimination or the identification of a substance with a definite source. However, much research is still needed to assess the validity of the results in order to use them either in an operational prospect or in court. Through the isotopic study of black powders and ammonium nitrates, this research aims at evaluating the contribution of isotope ratio mass spectrometry to the investigation of explosives, both from a pre-blast and from a post-blast approach. More specifically, the goal of the research is to provide additional elements necessary to a valid interpretation of the results, when used in explosives investigation. This work includes a fundamental study on the variability of the isotopic profile of black powder and ammonium nitrate in both space and time. On one hand, the inter-variability between manufacturers and, particularly, the intra-variability within a manufacturer has been studied. On the other hand, the stability of the isotopic profile over time has been evaluated through the aging of these substances exposed to different environmental conditions. The second part of this project considers the applicability of this high-precision technology to traces and residues of explosives, taking account of the characteristics specific to the field, including their sampling, a probable isotopic fractionation during the explosion, and the interferences with the matrix of the site.

Study of the application of the high performance liquid chromatography-particle beam interface-mass spectrometry technique on some pesiticides and the reduction phenomena in the system

This work includes two major parts. The first part of the work concentrated on the studies of the application of the highperfonnance liquid chromatography-particle beam interface-mass spectrometry system of some pesticides. Factors that have effects on the detection sensitivity were studied. The linearity ranges and detection limits of ten pesticides are also given in this work. The second part of the work concentrated on the studies of the reduction phenomena of nitro compounds in the HPLC-PB-MS system. Direct probe mass spectrometry and gas chromatography-mass spectrometry techniques were also used in the work. Factors that have effects on the reduction of the nitro compounds were studied, and the possible explanation is proposed. The final part of this work included the studies of reduction behavior of some other compounds in the HPLC-PB-MS system, included in them are: quinones, sulfoxides, and sulfones.

Large scale identification of protein SUMOylation by mass spectrometry in HEK293 cells

Les fichiers qui accompagnent mon document sont des tableaux supplémentaires réalisés avec Excel (Microsoft Office), dans la version papier du mémoire ces fichiers sont sur un CD-ROM.

Assessment of meat authenticity using bioinformatics, targeted peptide biomarkers and high-resolution mass spectrometry

In recent years, we observed a significant increase of food fraud ranging from false label claims to the use of additives and fillers to increase profitability. Recently in 2013, horse and pig DNA were detected in beef products sold from several retailers. Mass spectrometry has become the workhorse in protein research and the detection of marker proteins could serve for both animal species and tissue authentication. Meat species authenticity will be performed using a well defined proteogenomic annotation, carefully chosen surrogate tryptic peptides and analysis using a hybrid quadrupole-Orbitrap mass spectrometer. Selected mammalian meat samples were homogenized, proteins were extracted and digested with trypsin. The samples were analyzed using a high-resolution mass spectrometer. The chromatography was achieved using a 30 minutes linear gradient along with a BioBasic C8 100 × 1 mm column at a flow rate of 75 µL/min. The mass spectrometer was operated in full-scan high resolution and accurate mass. MS/MS spectra were collected for selected proteotypic peptides. Muscular proteins were methodically analyzed in silico in order to generate tryptic peptide mass lists and theoretical MS/MS spectra. Following a comprehensive bottom-up proteomic analysis, we were able to detect and identify a proteotypic myoglobin tryptic peptide [120-134] for each species with observed m/z below 1.3 ppm compared to theoretical values. Moreover, proteotypic peptides from myosin-1, myosin-2 and -hemoglobin were also identified. This targeted method allowed a comprehensive meat speciation down to 1% (w/w) of undesired product.

Mass spectrometry in clinical proteomics - from the present to the future

MS is an important analytical tool in clinical proteomics, primarily in the disease-specific discovery identification and characterisation of proteomic biomarkers and patterns. MS-based proteomics is increasingly used in clinical validation and diagnostic method development. The latter departs from the typical application of MS-based proteomics by exchanging some of the high performance of analysis for the throughput, robustness and simplicity required for clinical diagnostics. Although conventional MS-based proteomics has become an important field in clinical applications, some of the most recent MS technologies have not yet been extensively applied in clinical proteomics. in this review, we will describe the current state of MS in clinical proteomics and look to the future of this field.

Development of a liquid chromatographic time-of-flight mass spectrometric method for the determination of unlabelled and deuterium-labelled alpha-tocopherol in blood components

A method is described for the analysis of deuterated and undeuterated alpha-tocopherol in blood components using liquid chromatography coupled to an orthogonal acceleration time-of-flight (TOF) mass spectrometer. Optimal ionisation conditions for undeuterated (d0) and tri- and hexadeuterated (d3 or d6) alpha-tocopherol standards were found with negative ion mode electrospray ionisation. Each species produced an isotopically resolved single ion of exact mass. Calibration curves of pure standards were linear in the range tested (0-1.5 muM, 0-15 pmol injected). For quantification of d0 and d6 in blood components following a standard solvent extraction, a stable-isotope-labelled internal standard (d3-alpha-tocopherol) was employed. To counter matrix ion suppression effects, standard response curves were generated following identical solvent extraction procedures to those of the samples. Within-day and between-day precision were determined for quantification of d0- and d6-labelled alpha-tocopherol in each blood component and both averaged 3-10%. Accuracy was assessed by comparison with a standard high-performance liquid chromatography (HPLC) method, achieving good correlation (r(2) = 0.94), and by spiking with known concentrations of alpha-tocopherol (98% accuracy). Limits of detection and quantification were determined to be 5 and 50 fmol injected, respectively. The assay was used to measure the appearance and disappearance of deuterium-labelled alpha-tocopherol in human blood components following deuterium-labelled (d6) RRR-alpha-tocopheryl acetate ingestion. The new LC/TOFMS method was found to be sensitive, required small sample volumes, was reproducible and robust, and was capable of high throughput when large numbers of samples were generated. Copyright (C) 2003 John Wiley Sons, Ltd.

Characterization of the Salmonella typhimurium proteome by semi-automated two dimensional HPLC-mass spectrometry: detection of proteins implicated in multiple antibiotic resistance

The proteome of Salmonella enterica serovar Typhimurium was characterized by 2-dimensional HPLC mass spectrometry to provide a platform for subsequent proteomic investigations of low level multiple antibiotic resistance (MAR). Bacteria (2.15 +/- 0.23 x 10(10) cfu; mean +/- s.d.) were harvested from liquid culture and proteins differentially fractionated, on the basis of solubility, into preparations representative of the cytosol, cell envelope and outer membrane proteins (OMPs). These preparations were digested by treatment with trypsin and peptides separated into fractions (n = 20) by strong cation exchange chromatography (SCX). Tryptic peptides in each SCX fraction were further separated by reversed-phase chromatography and detected by mass spectrometry. Peptides were assigned to proteins and consensus rank listings compiled using SEQUEST. A total of 816 +/- 11 individual proteins were identified which included 371 +/- 33, 565 +/- 15 and 262 +/- 5 from the cytosolic, cell envelope and OMP preparations, respectively. A significant correlation was observed (r(2) = 0.62 +/- 0.10; P < 0.0001) between consensus rank position for duplicate cell preparations and an average of 74 +/- 5% of proteins were common to both replicates. A total of 34 outer membrane proteins were detected, 20 of these from the OMP preparation. A range of proteins (n = 20) previously associated with the mar locus in E. coli were also found including the key MAR effectors AcrA, TolC and OmpF.

A decade of plant proteomics and mass spectrometry: translation of technical advancements to food security and safety issues

Tremendous progress in plant proteomics driven by mass spectrometry (MS) techniques has been made since 2000 when few proteomics reports were published and plant proteomics was in its infancy. These achievements include the refinement of existing techniques and the search for new techniques to address food security, safety, and health issues. It is projected that in 2050, the world’s population will reach 9–12 billion people demanding a food production increase of 34–70% (FAO, 2009) from today’s food production. Provision of food in a sustainable and environmentally committed manner for such a demand without threatening natural resources, requires that agricultural production increases significantly and that postharvest handling and food manufacturing systems become more efficient requiring lower energy expenditure, a decrease in postharvest losses, less waste generation and food with longer shelf life. There is also a need to look for alternative protein sources to animal based (i.e., plant based) to be able to fulfill the increase in protein demands by 2050. Thus, plant biology has a critical role to play as a science capable of addressing such challenges. In this review, we discuss proteomics especially MS, as a platform, being utilized in plant biology research for the past 10 years having the potential to expedite the process of understanding plant biology for human benefits. The increasing application of proteomics technologies in food security, analysis, and safety is emphasized in this review. But, we are aware that no unique approach/technology is capable to address the global food issues. Proteomics-generated information/resources must be integrated and correlated with other omics-based approaches, information, and conventional programs to ensure sufficient food and resources for human development now and in the future.

Coupling Virus-Induced Gene Silencing to Exogenous Green Fluorescence Protein Expression Provides a Highly Efficient System for Functional Genomics in Arabidopsis and across All Stages of Tomato Fruit Development

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of ""sectoring"" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.

Characterization of O(2) ((1)Delta(g))-Derived Oxidation Products of Tryptophan: A Combination of Tandem Mass Spectrometry Analyses and Isotopic Labeling Studies

The fragmentation mechanisms of singlet oxygen [O(2) ((1)Delta(g))]-derived oxidation products of tryptophan (W) were analyzed using collision-induced dissociation coupled with (18)O-isotopic labeling experiments and accurate mass measurements. The five identified oxidized products, namely two isomeric alcohols (trans and cis WOH), two isomeric hydroperoxides (trans and cis WOOH), and N-formylkynurenine (FMK), were shown to share some common fragment ions and losses of small neutral molecules. Conversely, each oxidation product has its own fragmentation mechanism and intermediates, which were confirmed by (18)O-labeling studies. Isomeric WOH lost mainly H(2)O + CO, while WOOH showed preferential elimination of C(2)H(5)NO(3) by two distinct mechanisms. Differences in the spatial arrangement of the two isomeric WOHs led to differences in the intensities of the fragment ions. The same behavior was also found for trans and cis WOOH. FMK was shown to dissociate by a diverse range of mechanisms, with the loss of ammonia the most favored route. MS/MS analyses, (18)O-labeling, and H(2)(18)O experiments demonstrated the ability of FMK to exchange its oxygen atoms with water. Moreover, this approach also revealed that the carbonyl group has more pronounced oxygen exchange ability compared with the formyl group. The understanding of fragmentation mechanisms involved in O(2) ((1)Delta(g))-mediated oxidation of W provides a useful step toward the structural characterization of oxidized peptides and proteins. (J Am Soc Mass Spectrom 2009, 20, 188-197) (C) 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry

A theoretical and mass spectrometry study of the fragmentation of mycosporine-like amino acids

In the present study, the mycosporine-like amino acids (MAAs) were isolated from the marine red alga Gracilaria tenuistipitata and analysed by high-resolution accurate-mass sequential mass spectrometry (MSn). In addition to the proposed fragmentation mechanism based on the MSn analysis, it is clearly demonstrated that the elimination of mass 15 is a radical processes taking place at the methoxyl substituent of the double bond. This characteristic loss of a methyl radical was studied by theoretical calculations and the homolytic cleavage of the O-C bond is suggested to be dependent on the bond weakening. The protonation site of the MAAs was indicated by analysis of the Fukui functions and the relative Gibbs energies of the several possible protonated forms. (C) 2008 Elsevier B.V. All rights reserved.

Peat as a natural solid-phase for copper preconcentration and determination in a multicommuted flow system coupled to flame atomic absorption spectrometry

The physical and chemical characteristics of peat were assessed through measurement of pH, percentage of organic matter, cationic exchange capacity (CEC), elemental analysis, infrared spectroscopy and quantitative analysis of metals by ICP OES. Despite the material showed to be very acid in view of the percentage of organic matter, its CEC was significant, showing potential for retention of metal ions. This characteristic was exploited by coupling a peat mini-column to a flow system based on the multicommutation approach for the in-line copper concentration prior to flame atomic absorption spectrometric determination. Cu(II) ions were adsorbed at pH 4.5 and eluted with 0.50 mol L(-1) HNO(3). The influence of chemical and hydrodynamic parameters, such as sample pH, buffer concentration, eluent type and concentration, sample flow-rate and preconcentration time were investigated. Under the optimized conditions, a linear response was observed between 16 and 100 mu g L(-1), with a detection limit estimated as 3 mu g L(-1) at the 99.7% confidence level and an enrichment factor of 16. The relative standard deviation was estimated as 3.3% (n = 20). The mini-column was used for at least 100 sampling cycles without significant variation in the analytical response. Recoveries from copper spiked to lake water or groundwater as well as concentrates used in hemodialysis were in the 97.3-111 % range. The results obtained for copper determination in these samples agreed with those achieved by graphite furnace atomic absorption spectrometry (GFAAS) at the 95% confidence level. (C) 2009 Elsevier B.V. All rights reserved.