Glyco-oxidation is linked to the pathophysiology of diabetes and diabetic complications. The process of glyco-oxidation generates reactive dicarbonyls, which form adducts on arginine residues in distributions throughout the proteome that are site-specific depending on the protein microenvironment. Dicarbonyl adducts are thus markers for glyco-oxidative stress. Various approaches using mass spectrometry permits the identification, localization, and quantification of these dicarbonyl adducts. Using MG as a model dicarbonyl, a shotgun proteomics approach identified the sites for modification of major plasma proteins. Thirty five sites on seven abundant plasma proteins were found, and investigation into the microenvironment surrounding the target arginine sites revealed a neighboring charged residue motif where adjacent residues were either negatively or positively charged. One of the sites identified was R257 in HSA, which is located in the important drug binding site I. We validated drug site I as a target for MG modification by the adaptation of two assays to monitor the effect of MG modification. MG significantly decreases the rate of hydrolysis of PGE2 in drug site I, and induces the displacement of prodan from drug site I. Molecular modeling of warfarin docking at drug site I with the MG-modified R257 resulted in significantly decreased binding and change in binding orientation. The oxidation products of susceptible residues methionine, tryptophan, and cysteine were evaluated using MRM of oxidized HSA peptides. Oxidation of methionine gave the M+16 single oxidized product, and M329 in HSA was the most responsive site. Oxidation of the sole W214 tryptophan produced the W+32 double oxidation product, and oxidation of C34 produced the C+48 triple oxidation product. MG, 3DG, and glucosone were evaluated for propensity to modify 12 HSA sites based on MRM of dicarbonyl modified HSA. Dicarbonyl modification was independent of arginine solvent accessibility. In a clinical study using nephropathy as an endpoint, sites of oxidation and modification of HSA by MG, 3DG, and glucosone were quantified by MRM. The most important variable among diabetic subjects was metformin use, and subjects taking metformin had significantly reduced markers for glyco-oxidation. These findings may be useful in the development of new diabetes therapies that aim to ameliorate glyco-oxidative stress.

Le nombre croissant de médicaments protéiques utilisés en thérapeutique a créé des besoins dans le domaine de leur quantification, principalement dans le plasma, un milieu de composition protéique complexe. Le dosage, essentiel aux études pharmacocinétique/pharmacodynamique, ainsi qu’à l’optimisation de ces traitements, est compliqué par la nature protéique de ces médicaments et par les faibles concentrations auxquelles ils sont attendus dans ces milieux complexes. La méthodologie proposée se démarque des méthodes de dosage usuelles par son caractère universel. Elle fait appel à la spectrométrie de masse adaptée à la quantification des protéines grâce à l’utilisation d’un marquage isotopique différentiel des peptides : après enrichissement et protéolyse, l’échantillon à doser est marqué sur les lysines par la version légère d’un réactif de dérivation. En parallèle, les peptides de la protéine médicament pure marqués par la version lourde du réactif, servent d’étalon interne. La possibilité de quantifier la protéine à partir de plusieurs de ses peptides améliore la fiabilité du dosage. Appliquée à l’epoetin beta aux concentrations attendues en thérapeutique (autour de 0,5 femtomole/µL de plasma), la stratégie proposée permet de situer la limite de quantification à environ 50 attomoles d’epoetin beta/µL de plasma avec une méthodologie de spectrométrie de masse nano-LC-ESI-Q-TRAP fonctionnant en mode MRM. Pour étendre l’universalité de cette approche au champ des protéines médicaments pégylées, une seconde molécule a été étudiée. Il s’agit de l’interféron alfa-2b pégylé qui a permis de mettre en place une stratégie d’extraction spécifique du médicament utilisant sa pégylation. / The growing number of therapeutic proteins has created needs in the field of their quantification, mainly in plasma, which is a complex protein environment. Quantitative analysis of these proteins is essential for pharmacokinetics/pharmacodynamics studies, and for the optimization of treatments. However, the nature itself of the analyte and the low concentrations that are expected in plasma complicate the quantitative analysis. The proposed methodology differs from usual methods on its universal applicability. It relies on mass spectrometry adapted to the quantification of proteins by using peptides differential isotope labelling : after enrichment and proteolysis, the therapeutic protein and the plasmatic proteins are labelled on lysine residues by the light reagent. In parallel, peptides of the pure therapeutic protein, labelled by heavy version of reagent, are used as internal standard. The ability to quantify the protein with several of its peptides improves the reliability of the analysis. When applied to epoetin beta at expected therapeutic concentrations (about 0.5 femtomole/µL of plasma), the proposed strategy leads to a quantification limit close to 50 attomoles of epoetin beta/µL plasma, with a nano-LC-ESI-Q-TRAP mass spectrometry methodology operating in MRM. To extend the universal character of this approach to the field of pegylated protein drugs, a second therapeutic protein model has been studied. This model is a pegylated interferon alfa-2b which allowed developing a strategy for specific extraction of the drug relying on its pegylation.

Les cytochromes P450 (CYPs) jouent un rôle essentiel dans le métabolisme oxydatif de nombreux composés endogènes et exogènes. L’expression de CYPs est extrêmement variable en fonction de facteurs physiopathologiques, génétiques et environnementaux. Le métabolisme des xénobiotiques par les CYPs dépend en partie de la nature, de la quantité et de l’activité d’isoformes des CYPs impliqués. L'analyse quantitative de l'expression de CYP dans les organes du métabolisme, tels que le foie, sont d'une importance particulière étant donné que la biotransformation réalisée par les CYPs est souvent un facteur critique qui affecte l'efficacité, la disponibilité et la toxicité des médicaments chez l'homme. La technique actuelle de dosage la plus courante est l’immunoquantification par Western Blot. Cette technique est limitée par la disponibilité et la spécificité de l'anticorps. Les techniques de protéomique par spectrométrie de masse, permettant d’analyser de très faibles quantités de protéines en mélange, sont les méthodes de choix pour l’identification et la quantification des CYPs dans différents organes. Nous avons développé et validé une méthode pour doser 6 CYPs (1A2, 2C9, 2D6, 2J2, 3A4 et 3A5) par spectrométrie de masse en couplage chromatographique. Cette méthode, simple, rapide de sensibilité satisfaisante et peu coûteuse, a été validée dans différents types de matrices biologiques (lignées cellulaires hépatiques et neuronales, baculosomes). Ensuite, elle a été appliquée à grande échelle pour l’analyse de 50 foies humains (microsomes et mitochondries) afin d’étudier la relation phénotype/génotype pour les CYPs. Cette méthode pourra être appliquée à d’autres CYPS, est un outil utile qui permettra d’améliorer la compréhension et la prédiction pharmacocinétique et toxique de médicaments et d’autres produits chimiques. / Cytochromes P450 (CYPs) play a key role in the oxidative metabolism of many endogenous and exogenous compounds. The expression of CYPs is extremely variable depending on patho-physiological, genetic and environmental factors. The metabolism of xenobiotics by CYPs depends on the nature the quantity and the activity of CYP isoforms involved. Quantitative analysis of CYP expression in organs such as liver, are of particular importance since the biotransformation performed by CYPs is often a critical factor that affects the efficiency, availability and drug toxicity in humans. The most common technique is the immune-quantitation (Western Blot). This technique is limited by the availability and specificity of the antibody. Mass spectrometry-based proteomics, able to analyze very small amounts of protein in a mixture, are the methods of choice for identification and quantification of CYPs in different organs. We developed and validated a method for dosing 6 CYPs (1A2, 2C9, 2D6, 2J2, 3A4 and 3A5) by liquid chromatography coupled with mass spectrometry. This simple, rapid, low-cost method has an adequate sensitivity, and has been validated in different types of biological matrices (liver and neuronal cell lines, baculosomes). It has been applied at large-scale to analyze these 6 CYPs in 50 human livers samples (microsomes and mitochondria) to study the phenotype/genotype relationship. This method, which could easily be applied to other CYPs, provides an important tool to improve the understanding and prediction of pharmacokinetics and toxicity profile of drugs and other chemicals.

The World Health Organization has called stress-related illness “the health epidemic of the 21st century.” While the biochemical pathways associated with the acute stress response are well-characterized, many of the pathways behave differently under conditions of chronic stress. The purpose of this project is to apply high-sensitivity mass spectrometry (MS)-based targeted and untargeted metabolomics approaches to generate new insights into the biochemical processes and pathways associated with the chronic stress response, and potential mechanisms by which chronic stress produces adverse health effects. Chapter 1 describes the application of sets of targeted and untargeted metabolomics approaches to analyze serum samples from a human epigenetic model of chronic stress in order to identify potential targets for further analysis. To test the resulting hypothesis that oxidative stress is a key feature of chronic stress, a new targeted multiple reaction monitoring (MRM)-MS assay was developed for the accurate quantitation of aldehyde products of lipid peroxidation, as described in Chapter 2. In Chapter 3, the validated method for quantitation of malondialdehyde (MDA) was t applied to mouse plasma samples from a model of chronic social defeat stress to determine whether animals exposed to psychosocial stress show increases in oxidative stress. Mouse plasma samples from this model were also analyzed by untargeted metabolomics using Fourier-transform (FT)-MS to identify other important metabolite features, particularly those that overlap with metabolites identified in the human epigenetic model. Analysis of metabolomic data from two very different models of chronic stress supports the consistent detection of a metabolomic phenotype for chronic stress that is characterized by the dysregulation of energy metabolism associated with decreased concentrations of diacyl-phospholipids in blood. Increased blood concentrations of fatty acids, carnitines, acylcarnitines, and ether phospholipids were also observed. In addition to metabolites associated with energy metabolism, chronic stress also significantly influenced metabolites associated with amino acid metabolism and cell death. This characteristic pattern of differences in metabolite concentrations was observed in the plasma of mice exposed to chronic social defeat stress, irrespective of whether or not they displayed outward signs of a chronic stress response; In fact, mice that were “resilient” to the behavioural effects of chronic social defeat stress displayed an exaggerated phenotype over mice that showed depressive-like symptoms following chronic stress exposure. This may suggest that the observed changes in fatty acid composition are protective against stress. However, changes in fatty acid composition are also known to be associated with a wide variety of pathologies including heart disease, neurodegenerative diseases, and mood disorders, so the lipidomic changes associated with chronic stress may also contribute to its health impact. Overall, the results provide further evidence that changes in energy metabolism are a central part of allostatic adaptation to chronic stress. / Graduate / 0487 / csobsey@gmail.com

Protein tyrosine nitration (PTN) is a posttranslational modification resulting from oxidative/nitrosative stress that has been implicated in a wide variety of disease states. Characterization of PTN is challenging due to several factors including its low abundance in a given proteome, preferential site modification, multiple target site proximity within unique peptide sequences, and analytical method and instrument limitations. Current analytical techniques are insufficiently sensitive to identify endogenous nitration sites without incorporation of either nitrotyrosine or target protein enrichment. However, enrichment proficiency can also be inadequate. Chemical derivatization of the nitro- moiety can be incomplete or result in undesirable byproduct formation, while immunoaffinity proficiency is contingent upon antibody specificity. To overcome analytical method and enrichment deficiencies, we aimed to develop a comprehensive nitroproteome-specific workflow using molecular methods combined with mass spectrometry. Our approach was to systematically address all relevant factors contributing to PTN such as primary sequence, protein conformation, solvent accessibility, and nitrating agent concentration. Our ultimate goal was to increase mass spectrometric sensitivity for PTN identification. All putative nitroprotein/nitropeptide identifications were then subjected to rigorous validation by either manual spectrum analyses or peptide synthesis. We further developed MS methods for quantitation of nitropeptides from complex mixtures with minimal sample processing. Successful application of our nitroproteome-specific mass spectrometric workflow is expected to provide powerful tools for comprehensive PTN investigation that will elucidate its role in the onset and progression of a variety of disease states as well as facilitate discovery of therapeutic targets.

An optimized method for the chiral resolution of enantiomers of amino acids in bacterial supernatants is reported. This LC-MS/MS method is performed using a chiral Teichoplanin LC column and does not require sample clean up or chemical derivitization. This method allows for the determination of the relative amounts of the D and L enantiomers of 20 proteinogenic amino acids. The detection limits and response factors for the 20 amino acids were determined. Calibrations over three orders of magnitude showed least squares coefficient values (R^2) greater than 0.996 for eighty percent of the amino acids and greater than 0.992 for the remainder. The amino acids and their enantiomers were identified based on their retention times and their unique Multiple Reaction Monitoring (MRM) transitions for each amino acid. L-Aspartic acid-2,3,3-d3 was used as the internal standard. Cultures of Sinorhizobium meliloti (a nitrogen-fixing soil bacterium) were grown on minimal media; thus, all amino acids were biosynthesized by the bacterium. After centrifugation, supernatants were freeze dried, reconstituted in a small volume of methanol/water with internal standard and injected onto the LC column. The amino acids detected in the bacterial supernatant and the concentrations of the enantiomers were reported as the L and D isomers respectively: arginine [L, 12.6 ± 3.1 μg/L; D, 10.1 ± 3.2 μg/L], serine [L, 7.2 ± 1.16 μg/L; D, n.d.], threonine [L, n.d.; D, 11.2 ± 2.7 μg/L] and valine [L, 15.5 ± 4.3 μg/L; D, 11.3 ± 3.7 μg/L], where the term n.d. means below detection limit. The limits for detection for all amino acids ranged from 1.3 μg/L - 5.1 μg/L. In media with no added phosphate, the amino acid profiles changed somewhat under these stress conditions. Arginine was no longer detected while alanine and proline were now observed; the concentrations of the amino acids were: alanine [L, 7.7 ± 1.2 μg/L; D, 13.4 ± 2.5 μg/L], proline [L, n.d.; D, 8.63 ± 1.3 μg/L], serine [L, 7.6 ± 1.2 μg/L; D, n.d.], threonine [L, n.d.; D, 10.2 ± 3.2 μg/L] and valine [L, 11.6 ± 2.3 μg/L; D, 10.1 ± 3.1 μg/L]. These data represent the mean values of three independent bacterial growth experiments conducted over a 3 month period; the data came from the analysis of five separate aliquots from each growth experiment. The percent standard deviation for these data ranged from 15% to 33% and averaged 24%. Under both the normal and stressed growth conditions of S. meliloti produced the L enantiomer of serine, the D enantiomer of threonine and racemic valine. While racemic arginine was observed under normal growth conditions, levels were below detection under stressed conditions; under stress conditions only the D enantiomer of proline was observed while alanine was found in 1:2, L:D ratio. / Thesis / Master of Science (MSc)

A biotecnologia é definida como qualquer aplicação tecnológica que utilize sistemas biológicos, organismos vivos, ou seus derivados, para fabricar ou modificar produtos ou processos para alguma utilização específica (ORGANIZAÇÃO DAS NAÇÕES UNIDAS, 1992). Os produtos biotecnológicos devem atender certas especificações exigidas pela Agência Nacional de Vigilância Sanitária (ANVISA), Food and Drug Administration (FDA), European Medicine Agency (EMEA) e Wood Health Organization (WHO). Para isso, utilizam-se técnicas rotineiras aplicadas à pesquisa e análise de biomoléculas como Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western blot, Enzyme-Linked Immunosorbent Assay (ELISA) e espectrometria de massas (MS). A abordagem do Monitoramento de Reações Múltiplas (MRM) é considerada uma interessante alternativa aos ensaios imunoenzimáticos para caracterização e quantificação total de proteínas terapêuticas, sejam elas recombinantes ou não (KIM e DOYLE, 2010). Esta abordagem é baseada nos fundamentos da proteômica quantitativa pela utilização da cromatografia líquida acoplada à espectrometria de massas sequencial (LCMS/MS) cuja plataforma apresenta alta especificidade, sensibilidade e reprodutibilidade quantitativa em suas análises para detecção simultânea de várias regiões da estrutura proteica. Em vista disso, estabeleceu-se uma metodologia para quantificação do FVIII recombinante (FVIIIr) produzido pela linhagem celular humana Sk-Hep-1 ou do FVIII derivado do plasma (FVIIIdp), ambos utilizados no tratamento da Hemofilia A (HEMA). Para o estabelecimento da metodologia, toda uma estratégia de seleção e síntese química de peptídeos representativos das cadeias pesada e leve do FVIII humano foi empregada. Tais peptídeos obtidos foram utilizados como padrões das análises. Além disso, foi demonstrada a relação direta entre a quantificação total do FVIII com técnicas convencionais como ELISA, possibilitando a aplicação rotineira dessa abordagem para quantificação do FVIIIr e do FVIIIdp. / Biotechnology is defined as any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for a specific use (ORGANIZAÇÃO DAS NAÇÕES UNIDAS, 1992). Biotechnological products must meet certain specifications required by Agência Nacional de Vigilância Sanitária (ANVISA), Food and Drug Administration (FDA), European Medicine Agency (EMEA), and Wood Health Organization (WHO). For that routine techniques applied to research and analysis of biomolecules such Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western blot, Enzyme-Linked Immunosorbent Assay (ELISA), and mass spectrometry (MS) are utilized as quality control techniques. The MS-based approach termed Multiple Reaction Monitoring (MRM) is considered an interesting alternative to immunoassays for the total quantification and characterization of therapeutic proteins, whether recombinant or not (KIM e DOYLE, 2010). This approach is based on the fundamentals of quantitative proteomics and uses of liquid chromatography coupled to tandem spectrometric mass (LC-MS/MS) to obtain a highly specific, sensitive and reproducible analysis for quantification of multiple regions of a given protein structure. Here, we established a methodology for total quantification of recombinant FVIII (rFVIII) produced in Sk-Hep-1 human cell line or of plasma derived FVIII (pdFVIII), both used in the treatment of Hemophilia A (HEMA). For that, we adopted a strategy of selection and chemical peptide synthesis of representative peptides of the heavy and light chains of human FVIII, which were used as standards in the analysis. The quantitative MRM method developed here indicated a direct correlation with FVIII quantitative techniques such ELISA, which allows routine application of such approach for rFVIII and pdFVIII quantification.

A biotecnologia é definida como qualquer aplicação tecnológica que utilize sistemas biológicos, organismos vivos, ou seus derivados, para fabricar ou modificar produtos ou processos para alguma utilização específica (ORGANIZAÇÃO DAS NAÇÕES UNIDAS, 1992). Os produtos biotecnológicos devem atender certas especificações exigidas pela Agência Nacional de Vigilância Sanitária (ANVISA), Food and Drug Administration (FDA), European Medicine Agency (EMEA) e Wood Health Organization (WHO). Para isso, utilizam-se técnicas rotineiras aplicadas à pesquisa e análise de biomoléculas como Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western blot, Enzyme-Linked Immunosorbent Assay (ELISA) e espectrometria de massas (MS). A abordagem do Monitoramento de Reações Múltiplas (MRM) é considerada uma interessante alternativa aos ensaios imunoenzimáticos para caracterização e quantificação total de proteínas terapêuticas, sejam elas recombinantes ou não (KIM e DOYLE, 2010). Esta abordagem é baseada nos fundamentos da proteômica quantitativa pela utilização da cromatografia líquida acoplada à espectrometria de massas sequencial (LCMS/MS) cuja plataforma apresenta alta especificidade, sensibilidade e reprodutibilidade quantitativa em suas análises para detecção simultânea de várias regiões da estrutura proteica. Em vista disso, estabeleceu-se uma metodologia para quantificação do FVIII recombinante (FVIIIr) produzido pela linhagem celular humana Sk-Hep-1 ou do FVIII derivado do plasma (FVIIIdp), ambos utilizados no tratamento da Hemofilia A (HEMA). Para o estabelecimento da metodologia, toda uma estratégia de seleção e síntese química de peptídeos representativos das cadeias pesada e leve do FVIII humano foi empregada. Tais peptídeos obtidos foram utilizados como padrões das análises. Além disso, foi demonstrada a relação direta entre a quantificação total do FVIII com técnicas convencionais como ELISA, possibilitando a aplicação rotineira dessa abordagem para quantificação do FVIIIr e do FVIIIdp. / Biotechnology is defined as any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for a specific use (ORGANIZAÇÃO DAS NAÇÕES UNIDAS, 1992). Biotechnological products must meet certain specifications required by Agência Nacional de Vigilância Sanitária (ANVISA), Food and Drug Administration (FDA), European Medicine Agency (EMEA), and Wood Health Organization (WHO). For that routine techniques applied to research and analysis of biomolecules such Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western blot, Enzyme-Linked Immunosorbent Assay (ELISA), and mass spectrometry (MS) are utilized as quality control techniques. The MS-based approach termed Multiple Reaction Monitoring (MRM) is considered an interesting alternative to immunoassays for the total quantification and characterization of therapeutic proteins, whether recombinant or not (KIM e DOYLE, 2010). This approach is based on the fundamentals of quantitative proteomics and uses of liquid chromatography coupled to tandem spectrometric mass (LC-MS/MS) to obtain a highly specific, sensitive and reproducible analysis for quantification of multiple regions of a given protein structure. Here, we established a methodology for total quantification of recombinant FVIII (rFVIII) produced in Sk-Hep-1 human cell line or of plasma derived FVIII (pdFVIII), both used in the treatment of Hemophilia A (HEMA). For that, we adopted a strategy of selection and chemical peptide synthesis of representative peptides of the heavy and light chains of human FVIII, which were used as standards in the analysis. The quantitative MRM method developed here indicated a direct correlation with FVIII quantitative techniques such ELISA, which allows routine application of such approach for rFVIII and pdFVIII quantification.

Current screening tests for type 2 diabetes mellitus (T2DM) identify less than 50% of undiagnosed T2DM patients and provide no information about how the disease will develop in prediabetic patients. Here, twenty-nine protein glycation sites were quantified after tryptic digestion of plasma samples at the peptide level using tandem mass spectrometry and isotope-labelled peptides as internal standard. The glycation degrees were determined in three groups, i.e., 48 patients with a duration of T2DM exceeding ten years, 48 non-diabetic individuals matched for gender, BMI, and age, and 20 prediabetic men. In long-term controlled diabetic patients, 27 glycated peptides were detected at significantly higher levels, providing moderate diagnostic accuracies (ACCs) from 61 to 79%, allowing a subgrouping of patients in three distinct clusters. Moreover, a feature set of one glycated peptides and six established clinical parameters provided an ACC of 95%. The same number of clusters was identified in prediabetic males (ACC of 95%) using a set of eight glycation sites (mostly from serum albumin). All patients present in one cluster showed progression of prediabetic state or advanced towards diabetes in the following five years. Overall, the studied glycation sites appear to be promising biomarkers for subgrouping prediabetic patients to estimate their risk for the development of T2DM.

Ce travail de thèse rentre dans le cadre d’un projet de recherche appliquée mené à l’Institut National de Criminalistique et de Criminologie s’intitulant « développement d’un système intégral de détection et d’identification de résidus de tir » visant à doter le laboratoire de balistique chimique d’un outil performant pour la recherche et la caractérisation des résidus de tir aussi bien inorganiques qu’organiques. Comme nous l’expliquons dans le chapitre 1, l’analyse des résidus de tir est aujourd’hui essentiellement basée sur la mise en évidence par microscopie électronique à balayage des particules métalliques provenant de l’amorce. Cette pratique a des limites. Introductif, ce chapitre met en lumière la relation entre criminologie et criminalistique, donne un aperçu de la classification des armes et décrit les relations entre les armes de poing, les munitions, les résidus de tir et la criminalistique. La motivation de l’analyse de résidus organiques et plus particulièrement des stabilisants pour poudres propulsives y est aussi expliquée. Le but principal de ce travail étant le développement d’une méthode d’analyse des stabilisants par spectrométrie de masse en tandem couplée à la chromatographie liquide à haute performance (LC-MS/MS), ce premier chapitre aborde de façon détaillée le principe de fonctionnement d’un spectromètre de masse quadripolaire, qu’il soit avec simple quadripôle ou en tandem, et traite avec une attention particulière les aspects relatifs à la production d’ions, l’acquisition des données et la détection des ions. Le chapitre 2 est consacré aux résultats et discussions. Comme point de départ de tous les travaux ultérieurs, l’optimisation du fonctionnement du spectromètre et plus particulièrement la détermination des « ions précurseurs » et des « ions produits » y est traitée en détails. De plus, l’étude de la fragmentation des ions précurseurs y occupe une place prépondérante et conditionne l’établissement d’une méthode de détection et d’acquisition des données. Dans ce même chapitre sont successivement traités la mise au point de la méthode chromatographique pour l’analyse des stabilisants par LC-MS/MS, le couplage LC-MS/MS, la collecte et préparation des échantillons, dont l’extraction sur phase solide (SPE). Disposant des procédures de traitement des échantillons et d’une méthode LC-MS/MS, il nous a été possible de mener l’étude de faisabilité de l’analyse des stabilisants dans les échantillons prélevés sur les mains d’un tireur. Cette étude fait l’objet d’une proposition de publication jointe en annexe. Pour être exploitable en criminalistique, ce travail est complété par une étude sur les fréquences d’observation des stabilisants pour poudres propulsives sur les mains des non-tireurs. L’ensemble des résultats nous permet de dresser un bilan et proposer des perspectives décrites dans le chapitre 3. A cet égard, nous proposons de poursuivre les essais relatifs aux tests de persistance des résidus de stabilisant sur les mains des tireurs et d’entamer une étude sur la caractérisation des résidus de tir de la nitrocellulose. Du point de vue de l’exploitation des acquis de ce travail, nous suggérons que les méthodes et procédures développées soient mises à l’épreuve dans les affaires nécessitant l’analyse des prélèvements effectués sur les vêtements d’un suspect ou d’une victime. Cette mise à l’épreuve permettra de comparer l’efficacité de la méthode LC-MS/MS à celle de la technique SEM/EDX habituellement utilisée. Le chapitre 4 est consacré aux matériels et méthodes détaillant notamment les procédures de collectes et de traitement des échantillons, les paramètres de fonctionnement du spectromètre de masse et les méthodes analytiques. <br> <br> This thesis deals with a research project entitled "Development of an integral system of detection and identification of gunshot residues”, carried out at the Belgian National Institute of Criminalistics and Criminology (NICC). This project aims at providing the Laboratory of Chemical Ballistics, a powerful tool to detect and identify organic gunshot residues. For instance, the analyses of gunshot residues is presently performed mainly for characterizing metallic particles originating from the primer, using scanning electron microscopy coupled to energy dispersive X-ray microanalysis (SEM-EDX). However this technique has its limits and the goal of the thesis was the development of a method for the analysis of the stabilizers (and their derivatives) present in propellant powders with the use of tandem mass spectrometry coupled to high performance liquid chromatography (LC-MS/MS). The introduction highlights the relations between Criminology and Criminalistics; it gives an outline of the classification of weapons, and describes how handguns, ammunitions, gunshot residues and Criminalistics can be linked when investigating criminal cases. The motivation of the analysis of organic gunshot residues and in particular the propellant powder stabilizers is also explained. Chapter 1 deals in detail with the working principle of a quadrupole mass spectrometer, whether it is with a single quadrupole or composed of a tandem mass spectrometer. Important aspects connected with the ion production, the data acquisition and the ion detection are also considered. Chapter 2 presents the results and discussions. It begins with the optimization of the operating conditions of the spectrometer, and focuses more specifically on the determination of “precursor ions" and "daughter ions”. The study of the precursor ion is more important because it is required for the choice of parameters involved in the data acquisition mode. The development of a liquid chromatography (LC) method and the procedures for collecting and preparing the samples are explained. Moreover, Chapter 2 summarizes the results of a feasibility study of the analyses of stabilizers in samples collected from the hands of a shooter. This study was published in a peer reviewed journal [1]. A complementary study on the frequencies of observation of stabilizers on the hands of non-shooter persons is reported at the end the Chapter. The results allow also proposing perspectives which are described in Chapter 3. In this respect, the characterization of nitrocellulose residues and the study of the persistence of stabilizer residues on the hands of shooters should be undertaken. From the practical point of view, the developed methods and procedures should be tested in real cases which involve the analyses of the samples taken from the clothing of suspects and/or victims. These tests should allow an assessment of the LC-MS/MS method compared to the efficiency of the SEM/EDX technique currently used. Chapter 4 is dedicated to the materials and methods; the sample collection and treatment procedures, as well as the working parameters of the mass spectrometer and the analytical methods are explained in detail. [1]. Désiré Laza, Ph.D.; Bart Nys, Ph.D.; Jan De Kinder, Ph.D.; Andrée Kirsch - De Mesmaeker, Ph.D.; and Cécile Moucheron, Ph.D. Development of a Quantitative LC-MS/MS Method for the Analysis of Common Propellant Powder Stabilizers in Gunshot Residue. J. Forensic Sci, July 2007, Vol. 52, N° 4, 842-850.