Development of Imaging Mass Spectrometry Analysis of Lipids in Biological and Clinically Relevant Applications

La spectrométrie de masse mesure la masse des ions selon leur rapport masse sur charge. Cette technique est employée dans plusieurs domaines et peut analyser des mélanges complexes. L’imagerie par spectrométrie de masse (Imaging Mass Spectrometry en anglais, IMS), une branche de la spectrométrie de masse, permet l’analyse des ions sur une surface, tout en conservant l’organisation spatiale des ions détectés. Jusqu’à présent, les échantillons les plus étudiés en IMS sont des sections tissulaires végétales ou animales. Parmi les molécules couramment analysées par l’IMS, les lipides ont suscité beaucoup d'intérêt. Les lipides sont impliqués dans les maladies et le fonctionnement normal des cellules; ils forment la membrane cellulaire et ont plusieurs rôles, comme celui de réguler des événements cellulaires. Considérant l’implication des lipides dans la biologie et la capacité du MALDI IMS à les analyser, nous avons développé des stratégies analytiques pour la manipulation des échantillons et l’analyse de larges ensembles de données lipidiques. La dégradation des lipides est très importante dans l’industrie alimentaire. De la même façon, les lipides des sections tissulaires risquent de se dégrader. Leurs produits de dégradation peuvent donc introduire des artefacts dans l’analyse IMS ainsi que la perte d’espèces lipidiques pouvant nuire à la précision des mesures d’abondance. Puisque les lipides oxydés sont aussi des médiateurs importants dans le développement de plusieurs maladies, leur réelle préservation devient donc critique. Dans les études multi-institutionnelles où les échantillons sont souvent transportés d’un emplacement à l’autre, des protocoles adaptés et validés, et des mesures de dégradation sont nécessaires. Nos principaux résultats sont les suivants : un accroissement en fonction du temps des phospholipides oxydés et des lysophospholipides dans des conditions ambiantes, une diminution de la présence des lipides ayant des acides gras insaturés et un effet inhibitoire sur ses phénomènes de la conservation des sections au froid sous N2. A température et atmosphère ambiantes, les phospholipides sont oxydés sur une échelle de temps typique d’une préparation IMS normale (~30 minutes). Les phospholipides sont aussi décomposés en lysophospholipides sur une échelle de temps de plusieurs jours. La validation d’une méthode de manipulation d’échantillon est d’autant plus importante lorsqu’il s’agit d’analyser un plus grand nombre d’échantillons. L’athérosclérose est une maladie cardiovasculaire induite par l’accumulation de matériel cellulaire sur la paroi artérielle. Puisque l’athérosclérose est un phénomène en trois dimension (3D), l'IMS 3D en série devient donc utile, d'une part, car elle a la capacité à localiser les molécules sur la longueur totale d’une plaque athéromateuse et, d'autre part, car elle peut identifier des mécanismes moléculaires du développement ou de la rupture des plaques. l'IMS 3D en série fait face à certains défis spécifiques, dont beaucoup se rapportent simplement à la reconstruction en 3D et à l’interprétation de la reconstruction moléculaire en temps réel. En tenant compte de ces objectifs et en utilisant l’IMS des lipides pour l’étude des plaques d’athérosclérose d’une carotide humaine et d’un modèle murin d’athérosclérose, nous avons élaboré des méthodes «open-source» pour la reconstruction des données de l’IMS en 3D. Notre méthodologie fournit un moyen d’obtenir des visualisations de haute qualité et démontre une stratégie pour l’interprétation rapide des données de l’IMS 3D par la segmentation multivariée. L’analyse d’aortes d’un modèle murin a été le point de départ pour le développement des méthodes car ce sont des échantillons mieux contrôlés. En corrélant les données acquises en mode d’ionisation positive et négative, l’IMS en 3D a permis de démontrer une accumulation des phospholipides dans les sinus aortiques. De plus, l’IMS par AgLDI a mis en évidence une localisation différentielle des acides gras libres, du cholestérol, des esters du cholestérol et des triglycérides. La segmentation multivariée des signaux lipidiques suite à l’analyse par IMS d’une carotide humaine démontre une histologie moléculaire corrélée avec le degré de sténose de l’artère. Ces recherches aident à mieux comprendre la complexité biologique de l’athérosclérose et peuvent possiblement prédire le développement de certains cas cliniques. La métastase au foie du cancer colorectal (Colorectal cancer liver metastasis en anglais, CRCLM) est la maladie métastatique du cancer colorectal primaire, un des cancers le plus fréquent au monde. L’évaluation et le pronostic des tumeurs CRCLM sont effectués avec l’histopathologie avec une marge d’erreur. Nous avons utilisé l’IMS des lipides pour identifier les compartiments histologiques du CRCLM et extraire leurs signatures lipidiques. En exploitant ces signatures moléculaires, nous avons pu déterminer un score histopathologique quantitatif et objectif et qui corrèle avec le pronostic. De plus, par la dissection des signatures lipidiques, nous avons identifié des espèces lipidiques individuelles qui sont discriminants des différentes histologies du CRCLM et qui peuvent potentiellement être utilisées comme des biomarqueurs pour la détermination de la réponse à la thérapie. Plus spécifiquement, nous avons trouvé une série de plasmalogènes et sphingolipides qui permettent de distinguer deux différents types de nécrose (infarct-like necrosis et usual necrosis en anglais, ILN et UN, respectivement). L’ILN est associé avec la réponse aux traitements chimiothérapiques, alors que l’UN est associé au fonctionnement normal de la tumeur.

Identification of protein targets of nevirapine reactive metabolites using click chemistry and mass spectrometry-based differential proteomics

Abstract : Adverse drug reactions (ADRs) are undesirable effects caused after administration of a single dose or prolonged administration of drug or result from the combination of two or more drugs. Idiosyncratic drug reaction (IDR) is an adverse reaction that does not occur in most patients treated with a drug and does not involve the therapeutic effect of the drug. IDRs are unpredictable and often life-threatening. Idiosyncratic reaction is dependent on drug chemical characteristics or individual immunological response. IDRs are a major problem for drug development because they are usually not detected during clinical trials. In this study we focused on IDRs of Nevirapine (NVP), which is a non-nucleoside reverse transcriptase inhibitor used for the treatment of Human Immunodeficiency Virus (HIV) infections. The use of NVP is limited by a relatively high incidence of skin rash. NVP also causes a rash in female Brown Norway (BN) rats, which we use as animal model for this study. Our hypothesis is that idiosyncratic skin reactions associated with NVP treatment are due to post-translational modifications of proteins (e.g., glutathionylation) detectable by MS. The main objective of this study was to identify the proteins that are targeted by a reactive metabolite of Nevirapine in the skin. The specific objectives derived from the general objective were as follow: 1) To implement the click chemistry approach to detect proteins modified by a reactive NVP-Alkyne (NVP-ALK) metabolite. The purpose of using NVP-ALK was to couple it with Biotin using cycloaddition Click Chemistry reaction. 2) To detect protein modification using Western blotting and Mass Spectrometry techniques, which is important to understand the mechanism of NVP induced toxicity. 3) To identify the proteins using MASCOT search engine for protein identification, by comparing obtained spectrum from Mass Spectrometry with theoretical spectrum to find a matching peptide sequence. 4) To test if the drug or drug metabolites can cause harmful effects, as the induction of oxidative stress in cells (via protein glutathionylation). Oxidative stress causes cell damage that mediates signals, which likely induces the immune response. The results showed that Nevirapine is metabolized to a reactive metabolite, which causes protein modification. The extracted protein from the treated BN rats matched 10% of keratin, which implies that keratin was the protein targeted by the NVP-ALK.

Characterization of nucleic acids by tandem mass spectrometry - The second decade (2004-2013): From DNA to RNA and modified sequences

Nucleic acids play key roles in the storage and processing of genetic information, as well as in the regulation of cellular processes. Consequently, they represent attractive targets for drugs against gene-related diseases. On the other hand, synthetic oligonucleotide analogues have found application as chemotherapeutic agents targeting cellular DNA and RNA. The development of effective nucleic acid-based chemotherapeutic strategies requires adequate analytical techniques capable of providing detailed information about the nucleotide sequences, the presence of structural modifications, the formation of higher-order structures, as well as the interaction of nucleic acids with other cellular components and chemotherapeutic agents. Due to the impressive technical and methodological developments of the past years, tandem mass spectrometry has evolved to one of the most powerful tools supporting research related to nucleic acids. This review covers the literature of the past decade devoted to the tandem mass spectrometric investigation of nucleic acids, with the main focus on the fundamental mechanistic aspects governing the gas-phase dissociation of DNA, RNA, modified oligonucleotide analogues, and their adducts with metal ions. Additionally, recent findings on the elucidation of nucleic acid higher-order structures by tandem mass spectrometry are reviewed.

Sampling strategies for characterization of neuropeptides using mass spectrometry and functional implications into cell-cell signaling

In this dissertation, there are developed different analytical strategies to discover and characterize mammalian brain peptides using small amount of tissues. The magnocellular neurons of rat supraoptic nucleus in tissue and cell culture served as the main model to study neuropeptides, in addition to hippocampal neurons and mouse embryonic pituitaries. The neuropeptidomcis studies described here use different extraction methods on tissue or cell culture combined with mass spectrometry (MS) techniques, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). These strategies lead to the identification of multiple peptides from the rat/mouse brain in tissue and cell cultures, including novel compounds One of the goals in this dissertation was to optimize sample preparations on samples isolated from well-defined brain regions for mass spectrometric analysis. Here, the neuropeptidomics study of the SON resulted in the identification of 85 peptides, including 20 unique peptides from known prohormones. This study includes mass spectrometric analysis even from individually isolated magnocellular neuroendocrine cells, where vasopressin and several other peptides are detected. At the same time, it was shown that the same approach could be applied to analyze peptides isolated from a similar hypothalamic region, the suprachiasmatic nucleus (SCN). Although there were some overlaps regarding the detection of the peptides in the two brain nuclei, different peptides were detected specific to each nucleus. Among other peptides, provasopressin fragments were specifically detected in the SON while angiotensin I, somatostatin-14, neurokinin B, galanin, and vasoactive-intestinal peptide (VIP) were detected in the SCN only. Lists of peptides were generated from both brain regions for comparison of the peptidome of SON and SCN nuclei. Moving from analysis of magnocellular neurons in tissue to cell culture, the direct peptidomics of the magnocellular and hippocampal neurons led to the detection of 10 peaks that were assigned to previously characterized peptides and 17 peaks that remain unassigned. Peptides from the vasopressin prohormone and secretogranin-2 are attributed to magnocellular neurons, whereas neurokinin A, peptide J, and neurokinin B are attributed to cultured hippocampal neurons. This approach enabled the elucidation of cell-specific prohormone processing and the discovery of cell-cell signaling peptides. The peptides with roles in the development of the pituitary were analyzed using transgenic mice. Hes1 KO is a genetically modified mouse that lives only e18.5 (embryonic days). Anterior pituitaries of Hes1 null mice exhibit hypoplasia due to increased cell death and reduced proliferation and in the intermediate lobe, the cells differentiate abnormally into somatotropes instead of melanotropes. These previous findings demonstrate that Hes1 has multiple roles in pituitary development, cell differentiation, and cell fate. AVP was detected in all samples. Interestingly, somatostatin [92-100] and provasopressin [151-168] were detected in the mutant but not in the wild type or heterozygous pituitaries while somatostatin-14 was detected only in the heterozygous pituitary. In addition, the putative peptide corresponding to m/z 1330.2 and POMC [205-222] are detected in the mutant and heterozygous pituitaries, but not in the wild type. These results indicate that Hes1 influences the processing of different prohormones having possible roles during development and opens new directions for further developmental studies. This research demonstrates the robust capabilities of MS, which ensures the unbiased direct analysis of peptides extracted from complex biological systems and allows addressing important questions to understand cell-cell signaling in the brain.

Speciation analysis of Arsenic in fish and rice samples using liquid chromatography-inductively coupled plasma mass spectrometry

Chemical speciation in foodstuffs is of uttermost importance since it is nowadays recognized that both toxicity and bioavailability of an element depend on the chemical form in which the element is present. Regarding arsenic, inorganic species are classified as carcinogenic while organic arsenic, such as arsenobetaine (AsB) or arsenocholine (AsC), is considered less toxic or even non-toxic. Coupling a High Performance Liquid Chromatographer (HPLC) with an Inductively Coupled Plasma Mass Spectrometer (ICP-MS) combines the power of separation of the first with the selectivity and sensitivity of the second. The present work aims at developing a method, using HPLC-ICP-MS technique, to identify and quantify the chemical species of arsenic present in two food matrices, rice and fish. Two extraction methods, ultrasound and microwave, and different settings were studied. The best method was chosen based on recovery percentages. To ensure that no interconversion of species was occurring, individual spikes of each species of arsenic were made in both matrices and recovery rates were calculated. To guaranty accurate results reference material BCR-627 TUNA FISH, containing certified values for AsB and DMA, was analyzed. Chromatographic separation was achieved using an anion exchange column, HAMILTON-PRP X-100, which allowed to separate the four arsenic species for which standards were available (AsB, dimethylarsenic (DMA), arsenite (AsIII), arsenate (AsV). The mobile phase was chosen based on scientific literature and adjusted to laboratory conditions. Different gradients were studied. As a result we verified that the arsenic species present in both matrices were not the same. While in fish 90% of the arsenic present was in the form of arsenobetaine, in rice 80% of arsenic was present as DMA and 20% as inorganic arsenic.

Exploring complex chemical systems: insights from mass spectrometry, electrochemistry and continuous cell culture

The work presented herein covers a broad range of research topics and so, in the interest of clarity, has been presented in a portfolio format. Accordingly, each chapter consists of its own introductory material prior to presentation of the key results garnered, this is then proceeded by a short discussion on their significance. In the first chapter, a methodology to facilitate the resolution and qualitative assessment of very large inorganic polyoxometalates was designed and implemented employing ion-mobility mass spectrometry. Furthermore, the potential of this technique for ‘mapping’ the conformational space occupied by this class of materials was demonstrated. These claims are then substantiated by the development of a tuneable, polyoxometalate-based calibration protocol that provided the necessary platform for quantitative assessments of similarly large, but unknown, polyoxometalate species. In addition, whilst addressing a major limitation of travelling wave ion mobility, this result also highlighted the potential of this technique for solution-phase cluster discovery. The second chapter reports on the application of a biophotovoltaic electrochemical cell for characterising the electrogenic activity inherent to a number of mutant Synechocystis strains. The intention was to determine the key components in the photosynthetic electron transport chain responsible for extracellular electron transfer. This would help to address the significant lack of mechanistic understanding in this field. Finally, in the third chapter, the design and fabrication of a low-cost, highly modular, continuous cell culture system is presented. To demonstrate the advantages and suitability of this platform for experimental evolution investigations, an exploration into the photophysiological response to gradual iron limitation, in both the ancestral wild type and a randomly generated mutant library population, was undertaken. Furthermore, coupling random mutagenesis to continuous culture in this way is shown to constitute a novel source of genetic variation that is open to further investigation.

Characterizing neural tissues with mass spectrometry imaging via enhanced computational approaches

Neuropeptides affect the activity of the myriad of neuronal circuits in the brain. They are under tight spatial and chemical control and the dynamics of their release and catabolism directly modify neuronal network activity. Understanding neuropeptide functioning requires approaches to determine their chemical and spatial heterogeneity within neural tissue, but most imaging techniques do not provide the complete information desired. To provide chemical information, most imaging techniques used to study the nervous system require preselection and labeling of the peptides of interest; however, mass spectrometry imaging (MSI) detects analytes across a broad mass range without the need to target a specific analyte. When used with matrix-assisted laser desorption/ionization (MALDI), MSI detects analytes in the mass range of neuropeptides. MALDI MSI simultaneously provides spatial and chemical information resulting in images that plot the spatial distributions of neuropeptides over the surface of a thin slice of neural tissue. Here a variety of approaches for neuropeptide characterization are developed. Specifically, several computational approaches are combined with MALDI MSI to create improved approaches that provide spatial distributions and neuropeptide characterizations. After successfully validating these MALDI MSI protocols, the methods are applied to characterize both known and unidentified neuropeptides from neural tissues. The methods are further adapted from tissue analysis to be able to perform tandem MS (MS/MS) imaging on neuronal cultures to enable the study of network formation. In addition, MALDI MSI has been carried out over the timecourse of nervous system regeneration in planarian flatworms resulting in the discovery of two novel neuropeptides that may be involved in planarian regeneration. In addition, several bioinformatic tools are developed to predict final neuropeptide structures and associated masses that can be compared to experimental MSI data in order to make assignments of neuropeptide identities. The integration of computational approaches into the experimental design of MALDI MSI has allowed improved instrument automation and enhanced data acquisition and analysis. These tools also make the methods versatile and adaptable to new sample types.

High resolution mass spectrometry for quantitative analysis and untargeted screening of algal toxins in mussels and passive samplers

Measurement of marine algal toxins has traditionally focussed on shellfish monitoring while, over the last decade, passive sampling has been introduced as a complementary tool for exploratory studies. Since 2011, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been adopted as the EU reference method (No.15/2011) for detection and quantitation of lipophilic toxins. Traditional LC-MS approaches have been based on low-resolution mass spectrometry (LRMS), however, advances in instrument platforms have led to a heightened interest in the use of high-resolution mass spectrometry (HRMS) for toxin detection. This work describes the use of HRMS in combination with passive sampling as a progressive approach to marine algal toxin surveys. Experiments focused on comparison of LRMS and HRMS for determination of a broad range of toxins in shellfish and passive samplers. Matrix effects are an important issue to address in LC-MS; therefore, this phenomenon was evaluated for mussels (Mytilus galloprovincialis) and passive samplers using LRMS (triple quadrupole) and HRMS (quadrupole time-of-flight and Orbitrap) instruments. Matrix-matched calibration solutions containing okadaic acid and dinophysistoxins, pectenotoxin, azaspiracids, yessotoxins, domoic acid, pinnatoxins, gymnodimine A and 13-desmethyl spirolide C were prepared. Similar matrix effects were observed on all instruments types. Most notably, there was ion enhancement for pectenotoxins, okadaic acid/dinophysistoxins on one hand, and ion suppression for yessotoxins on the other. Interestingly, the ion selected for quantitation of PTX2 also influenced the magnitude of matrix effects, with the sodium adduct typically exhibiting less susceptibility to matrix effects than the ammonium adduct. As expected, mussel as a biological matrix, quantitatively produced significantly more matrix effects than passive sampler extracts, irrespective of toxin. Sample dilution was demonstrated as an effective measure to reduce matrix effects for all compounds, and was found to be particularly useful for the non-targeted approach. Limits of detection and method accuracy were comparable between the systems tested, demonstrating the applicability of HRMS as an effective tool for screening and quantitative analysis. HRMS offers the advantage of untargeted analysis, meaning that datasets can be retrospectively analysed. HRMS (full scan) chromatograms of passive samplers yielded significantly less complex data sets than mussels, and were thus more easily screened for unknowns. Consequently, we recommend the use of HRMS in combination with passive sampling for studies investigating emerging or hitherto uncharacterised toxins.

Plutonium release from Fukushima Daiichi fosters the need for more detailed investigations

The contamination of Japan after the Fukushima accident has been investigated mainly for volatile fission products, but only sparsely for actinides such as plutonium. Only small releases of actinides were estimated in Fukushima. Plutonium is still omnipresent in the environment from previous atmospheric nuclear weapons tests. We investigated soil and plants sampled at different hot spots in Japan, searching for reactor-borne plutonium using its isotopic ratio Pu-240/Pu-239. By using accelerator mass spectrometry, we clearly demonstrated the release of Pu from the Fukushima Daiichi power plant: While most samples contained only the radionuclide signature of fallout plutonium, there is at least one vegetation sample whose isotope ratio (0.381 +/- 0.046) evidences that the Pu originates from a nuclear reactor (Pu239+240 activity concentration 0.49 Bq/kg). Plutonium content and isotope ratios differ considerably even for very close sampling locations, e.g. the soil and the plants growing on it. This strong localization indicates a particulate Pu release, which is of high radiological risk if incorporated.

Tissue Proteomes: Quantitative Mass Spectrometry of Murine Liver and Ovarian Endometrioma

A human genome contains more than 20 000 protein-encoding genes. A human proteome, instead, has been estimated to be much more complex and dynamic. The most powerful tool to study proteins today is mass spectrometry (MS). MS based proteomics is based on the measurement of the masses of charged peptide ions in a gas-phase. The peptide amino acid sequence can be deduced, and matching proteins can be found, using software to correlate MS-data with sequence database information. Quantitative proteomics allow the estimation of the absolute or relative abundance of a certain protein in a sample. The label-free quantification methods use the intrinsic MS-peptide signals in the calculation of the quantitative values enabling the comparison of peptide signals from numerous patient samples. In this work, a quantitative MS methodology was established to study aromatase overexpressing (AROM+) male mouse liver and ovarian endometriosis tissue samples. The workflow of label-free quantitative proteomics was optimized in terms of sensitivity and robustness, allowing the quantification of 1500 proteins with a low coefficient of variance in both sample types. Additionally, five statistical methods were evaluated for the use with label-free quantitative proteomics data. The proteome data was integrated with other omics datasets, such as mRNA microarray and metabolite data sets. As a result, an altered lipid metabolism in liver was discovered in male AROM+ mice. The results suggest a reduced beta oxidation of long chain phospholipids in the liver and increased levels of pro-inflammatory fatty acids in the circulation in these mice. Conversely, in the endometriosis tissues, a set of proteins highly specific for ovarian endometrioma were discovered, many of which were under the regulation of the growth factor TGF-β1. This finding supports subsequent biomarker verification in a larger number of endometriosis patient samples.

Optimization of the Ion Source-Mass Spectrometry Parameters in Non-Steroidal Anti-Inflammatory and Analgesic Pharmaceuticals Analysis by a Design of Experiments Approach

The flow rates of drying and nebulizing gas, heat block and desolvation line temperatures and interface voltage are potential electrospray ionization parameters as they may enhance sensitivity of the mass spectrometer. The conditions that give higher sensitivity of 13 pharmaceuticals were explored. First, Plackett-Burman design was implemented to screen significant factors, and it was concluded that interface voltage and nebulizing gas flow were the only factors that influence the intensity signal for all pharmaceuticals. This fractionated factorial design was projected to set a full 2(2) factorial design with center points. The lack-of-fit test proved to be significant. Then, a central composite face-centered design was conducted. Finally, a stepwise multiple linear regression and subsequently an optimization problem solving were carried out. Two main drug clusters were found concerning the signal intensities of all runs of the augmented factorial design. p-Aminophenol, salicylic acid, and nimesulide constitute one cluster as a result of showing much higher sensitivity than the remaining drugs. The other cluster is more homogeneous with some sub-clusters comprising one pharmaceutical and its respective metabolite. It was observed that instrumental signal increased when both significant factors increased with maximum signal occurring when both codified factors are set at level +1. It was also found that, for most of the pharmaceuticals, interface voltage influences the intensity of the instrument more than the nebulizing gas flowrate. The only exceptions refer to nimesulide where the relative importance of the factors is reversed and still salicylic acid where both factors equally influence the instrumental signal. Graphical Abstract ᅟ.

CHARACTERIZATION OF POLYUBIQUITIN CHAINS BY MASS SPECTROMETRY

The work outlined in this dissertation will allow biochemists and cellular biologists to characterize polyubiquitin chains involved in their cellular environment by following a facile mass spectrometric based workflow. The characterization of polyubiquitin chains has been of interest since their discovery in 1984. The profound effects of ubiquitination on the movement and processing of cellular proteins depend exclusively on the structures of mono and polyubiquitin modifications anchored or unanchored on the protein within the cellular environment. However, structure-function studies have been hindered by the difficulty in identifying complex chain structures due to limited instrument capabilities of the past. Genetic mutations or reiterative immunoprecipitations have been used previously to characterize the polyubiquitin chains, but their tedium makes it difficult to study a broad ubiquitinome. Top-down and middle-out mass spectral based proteomic studies have been reported for polyubiquitin and have had success in characterizing parts of the chain, but no method to date has been successful at differentiating all theoretical ubiquitin chain isomers (ubiquitin chain lengths from dimer to tetramer alone have 1340 possible isomers). The workflow presented here can identify chain length, topology and linkages present using a chromatographic-time-scale compatible, LC-MS/MS based workflow. To accomplish this feat, the strategy had to exploit the most recent advances in top-down mass spectrometry. This included the most advanced electron transfer dissociation (ETD) activation and sensitivity for large masses from the orbitrap Fusion Lumos. The spectral interpretation had to be done manually with the aid of a graphical interface to assign mass shifts because of a lack of software capable to interpret fragmentation across isopeptide linkages. However, the method outlined can be applied to any mass spectral based system granted it results in extensive fragmentation across the polyubiquitin chain; making this method adaptable to future advances in the field.

Development of Imaging Mass Spectrometry Analysis of Lipids in Biological and Clinically Relevant Applications

La spectrométrie de masse mesure la masse des ions selon leur rapport masse sur charge. Cette technique est employée dans plusieurs domaines et peut analyser des mélanges complexes. L’imagerie par spectrométrie de masse (Imaging Mass Spectrometry en anglais, IMS), une branche de la spectrométrie de masse, permet l’analyse des ions sur une surface, tout en conservant l’organisation spatiale des ions détectés. Jusqu’à présent, les échantillons les plus étudiés en IMS sont des sections tissulaires végétales ou animales. Parmi les molécules couramment analysées par l’IMS, les lipides ont suscité beaucoup d'intérêt. Les lipides sont impliqués dans les maladies et le fonctionnement normal des cellules; ils forment la membrane cellulaire et ont plusieurs rôles, comme celui de réguler des événements cellulaires. Considérant l’implication des lipides dans la biologie et la capacité du MALDI IMS à les analyser, nous avons développé des stratégies analytiques pour la manipulation des échantillons et l’analyse de larges ensembles de données lipidiques. La dégradation des lipides est très importante dans l’industrie alimentaire. De la même façon, les lipides des sections tissulaires risquent de se dégrader. Leurs produits de dégradation peuvent donc introduire des artefacts dans l’analyse IMS ainsi que la perte d’espèces lipidiques pouvant nuire à la précision des mesures d’abondance. Puisque les lipides oxydés sont aussi des médiateurs importants dans le développement de plusieurs maladies, leur réelle préservation devient donc critique. Dans les études multi-institutionnelles où les échantillons sont souvent transportés d’un emplacement à l’autre, des protocoles adaptés et validés, et des mesures de dégradation sont nécessaires. Nos principaux résultats sont les suivants : un accroissement en fonction du temps des phospholipides oxydés et des lysophospholipides dans des conditions ambiantes, une diminution de la présence des lipides ayant des acides gras insaturés et un effet inhibitoire sur ses phénomènes de la conservation des sections au froid sous N2. A température et atmosphère ambiantes, les phospholipides sont oxydés sur une échelle de temps typique d’une préparation IMS normale (~30 minutes). Les phospholipides sont aussi décomposés en lysophospholipides sur une échelle de temps de plusieurs jours. La validation d’une méthode de manipulation d’échantillon est d’autant plus importante lorsqu’il s’agit d’analyser un plus grand nombre d’échantillons. L’athérosclérose est une maladie cardiovasculaire induite par l’accumulation de matériel cellulaire sur la paroi artérielle. Puisque l’athérosclérose est un phénomène en trois dimension (3D), l'IMS 3D en série devient donc utile, d'une part, car elle a la capacité à localiser les molécules sur la longueur totale d’une plaque athéromateuse et, d'autre part, car elle peut identifier des mécanismes moléculaires du développement ou de la rupture des plaques. l'IMS 3D en série fait face à certains défis spécifiques, dont beaucoup se rapportent simplement à la reconstruction en 3D et à l’interprétation de la reconstruction moléculaire en temps réel. En tenant compte de ces objectifs et en utilisant l’IMS des lipides pour l’étude des plaques d’athérosclérose d’une carotide humaine et d’un modèle murin d’athérosclérose, nous avons élaboré des méthodes «open-source» pour la reconstruction des données de l’IMS en 3D. Notre méthodologie fournit un moyen d’obtenir des visualisations de haute qualité et démontre une stratégie pour l’interprétation rapide des données de l’IMS 3D par la segmentation multivariée. L’analyse d’aortes d’un modèle murin a été le point de départ pour le développement des méthodes car ce sont des échantillons mieux contrôlés. En corrélant les données acquises en mode d’ionisation positive et négative, l’IMS en 3D a permis de démontrer une accumulation des phospholipides dans les sinus aortiques. De plus, l’IMS par AgLDI a mis en évidence une localisation différentielle des acides gras libres, du cholestérol, des esters du cholestérol et des triglycérides. La segmentation multivariée des signaux lipidiques suite à l’analyse par IMS d’une carotide humaine démontre une histologie moléculaire corrélée avec le degré de sténose de l’artère. Ces recherches aident à mieux comprendre la complexité biologique de l’athérosclérose et peuvent possiblement prédire le développement de certains cas cliniques. La métastase au foie du cancer colorectal (Colorectal cancer liver metastasis en anglais, CRCLM) est la maladie métastatique du cancer colorectal primaire, un des cancers le plus fréquent au monde. L’évaluation et le pronostic des tumeurs CRCLM sont effectués avec l’histopathologie avec une marge d’erreur. Nous avons utilisé l’IMS des lipides pour identifier les compartiments histologiques du CRCLM et extraire leurs signatures lipidiques. En exploitant ces signatures moléculaires, nous avons pu déterminer un score histopathologique quantitatif et objectif et qui corrèle avec le pronostic. De plus, par la dissection des signatures lipidiques, nous avons identifié des espèces lipidiques individuelles qui sont discriminants des différentes histologies du CRCLM et qui peuvent potentiellement être utilisées comme des biomarqueurs pour la détermination de la réponse à la thérapie. Plus spécifiquement, nous avons trouvé une série de plasmalogènes et sphingolipides qui permettent de distinguer deux différents types de nécrose (infarct-like necrosis et usual necrosis en anglais, ILN et UN, respectivement). L’ILN est associé avec la réponse aux traitements chimiothérapiques, alors que l’UN est associé au fonctionnement normal de la tumeur.

Organic compounds on comet 67P/Churyumov-Gerasimenko revealed by COSAC mass spectrometry

Comets harbor the most pristine material in our solar system in the form of ice, dust, silicates, and refractory organic material with some interstellar heritage. The evolved gas analyzer Cometary Sampling and Composition (COSAC) experiment aboard Rosetta's Philae lander was designed for in situ analysis of organic molecules on comet 67P/Churyumov-Gerasimenko. Twenty-five minutes after Philae's initial comet touchdown, the COSAC mass spectrometer took a spectrum in sniffing mode, which displayed a suite of 16 organic compounds, including many nitrogen-bearing species but no sulfur-bearing species, and four compounds-methyl isocyanate, acetone, propionaldehyde, and acetamide-that had not previously been reported in comets.

Development of new analytical procedures of isotope geochemistry by mass spectrometry. Application to the study of high-pressure metamorphic rocks from the Iberian Massif

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