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Mon travail de thèse a consisté à poursuivre le développement de l’Imagerie par Spectrométrie de Masse (IMS), à la fois sur le plan méthodologique mais aussi à travers des applications biologiques.Dans une première partie est exposé le développement d’une méthode de préparation des échantillons de très petite taille pour l’imagerie chimique, et plus particulièrement pour l’imagerie TOF-SIMS. Cette méthode a pu être validée en étudiant des molécules de la famille des flavonoïdes dans différents types de graines d’Arabidopsis thaliana ne mesurant que 400 nm de diamètre. La seconde partie, dédiée aux applications biologiques, est divisée en deux sections. La première section regroupe deux sujets où il était question de détecter et de localiser, à l’aide de l’imagerie TOF-SIMS, la molécule active d’une crème anti-acné dans des coupes de peaux de cadavre humain, ainsi qu’un retardateur de flamme bromé, le décabromodiphényl éther, dans ses tissus cibles chez le rat. Dans la seconde section, nous avons étudié, en imagerie TOF-SIMS et MALDI-TOF, l’absorption des lipides durant la digestion et enfin, avec l’aide d’outils d’analyse statistiques, nous avons comparé les profils lipidiques d’échantillons sains et atteins de la mucoviscidose chez un animal modèle de la maladie.À travers ces différents projets, nous avons pu conclure que les imageries par spectrométrie de masse TOF-SIMS et MALDI-TOF sont deux techniques complémentaires et qui, combinées à l’analyse statistique, peuvent être de puissants outils. / My PhD’s work consisted in continuing the development of Mass Spectrometry Imaging (MSI) methods, in terms of methodology improvements but also through biological applications.The first part concerned the development of a novel sample preparation method dedicated to very small objects for chemical imaging, particularly for TOF-SIMS imaging. This method has been validated by studying different types of flavonoids in from seeds of Arabidopsis thaliana, with a size of 400 nm only. The second part, dedicated to biological applications, is divided into two sections. The first section includes two projects where the goals was to detect and locate, using TOF-SIMS imaging, the active molecule of an anti-acne cream in human skin sections, and a brominated flame retardant, the decabromodiphenyl ether, in target tissues in rats. In the second section, we have studied by MALDI-TOF and TOF-SIMS imaging the lipid absorption during the digestion, and finally, with the help of statistical analysis tools, we compared lipid profiles of healthy samples versus those from cystic fibrosis samples in a model animal of the disease.Through these projects, we have concluded that MALDI-TOF and TOF-SIMS imaging are two complementary techniques, and, when they are combined with statistical analysis, they can be powerful tools.

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The interaction between the matrix and analyte molecules are studied with a high resolution MALDI imaging technique in an orthogonal-injection TOF mass spectrometer. The analyte incorporation and distribution patterns have been clearly demonstrated. Purified protein analytes were found to be homogeneously incorporated in large single crystals of DHB and sinapinic acid matrices, with no evidence for preferred crystal faces. Segregation of some species was observed and appeared to correlate with analyte hydrophobicity, and to a lesser extent analyte mass or mobility. The influence of incident laser parameters on sensitivity in MALDI has been investigated using orthogonal-injection time-of-flight (TOF) instruments. A qualitative comparison was first made between the beam profiles obtained with a N2 laser and a Nd:YAG laser using 2-m long optical fibers. The N2 laser gives better sensitivity, consistent with a more uniform fluence distribution and therefore better coverage of the N2 laser profile. Most of the difference disappears when a 30-m long fiber is used or when the fibers are twisted during irradiation to smooth out the fluence distribution. In more systematic measurements, the total integrated ion yield from a single spot (a measure of sensitivity) was found to increase rapidly with fluence to a maximum, and then saturate or decrease slightly. For a fluence near threshold, the integrated yield has a steep (cubic) dependence on the spot size, but the yield saturates at higher fluence for smaller spots. The area dependence is much weaker (close to linear) for fluence values above saturation, with the result that the highest integrated yields per unit area are obtained with the smallest spot sizes. Finally the detection properties of the MCP detector were studied with a hybrid MCP and CuBe venetian blind converter detector. The measurements show that the detection efficiency of the MCP drops with the increasing of ion mass and the decreasing of the ion energy. The secondary electron emission coefficient of the MCP shows a linear dependence on mass and a power law dependence on velocity (~ 3.2). No clear velocity threshold is observed for secondary electron emission. / May 2009

Bacterial research has been flourishing ever since 17th century. Bacteria play an important role in clinical microbiology, food microbiology and environmental microbiology. Therefore, the rising need to detect bacteria in clinical, food and environmental samples is emerging onto a craving research frenzy. There are some established methods to study bacteria, such as genetic techniques like PCR, culturing and RNA based analysis, but these require time, resources and labor. This thesis concentrates on highlighting the importance of MALDI-MS in rapid, direct, and simple detection of bacteria from food and clinical samples. The method is to enable detection of bacteria in real samples. First attempts were made to detect bacteria from yogurt sample, because as already known commercial yogurt samples contain bacteria. And yogurts were used in this study to demonstrate the capability of MALDI-MS technique to detect the bacteria directly by culture-free methods. Since the bacterial signals were quenched because of the strong milk peaks, we had to employ nanoparticles and ionic solution for affinity capture of the bacteria for bacterial detection. The results showed that addition of ionic solution and Ag nanoparticles lead to enhanced bacterial detection. The deterioration of the yogurt bacteria on improper storage ( at room temperature) and after extended storage in refrigerator beyond expiry date was studied using MALDI-MS. The day by day deterioration in the microbiological quality of the yogurt was also detected. A special selective media for growth of the yogurt bacteria, DifcoTM Lactobacilli MRS Broth (Agar) was used to quantify the bacteria in various experiments. MALDI-MS results were useful in understanding the shelf life and quality control on extended storage. The second part of this thesis concentrates on the use of MALDI-MS in clinical analysis. The major goal in this work was to use MALDI-MS to detect bacteria in mousy blood and urine. Phase I of this study involved detection of the bacteria by direct MALDI-MS in blood and urine samples spiked with bacteria. We call these experiments as in vitro experiments. Analysis using MALDI was a challenge since the blood peaks interfered with the bacterial peaks and subdued them. Ionic liquid is a pretty excellent extraction solvent when combined with SDME (Single-drop Microextraction). Just one drop of 1-butyl-3methylimidazolium hexafluorophosphate can extract bacteria from spiked samples. What¡¦s more, the LOD can be lower to ~105 cfu / mL using SDME with ionic liquid. It¡¦s definitely showing that 1-butyl-3methylimidazolium hexafluorophosphate, that is to say, the ionic liquid has good ability to trap pathogenic bacteria. This bacterium is injected into mouse and the growth kinetics of bacteria in vivo was studied as a function of time. Samples taken at specific time points were simultaneously analyzed using standard plate count method as well as MALDI-MS. Analysis using MALDI was a challenge since the blood peaks interfered with the bacterial peaks and subdued them. Therefore, we employ the use of silver and zinc oxide nanoparticles and ionic liquid.

Abstract Rapid and reliable identification of microorganisms is of paramount importance for advancing homeland security. Mass spectrometry is emerging as an effective bioanalytical tool having unique capabilities in handing complex mixtures. Matrix-assisted layser desorption / ionization time-of-flight mass spectrometry ¡]MALDI/TOF MS¡^is commonly used in the analysis of bio-molecules owing to its merits of high sensitivity, wide mass range, and ease-of-operation. The objective of the first part of the thesis is to develop an analysis method to characterize the genus, strains, and subspecies of the infections gastroenteritis bacteria by using MALDI/TOF MS. MALDI/TOF MS can be applied to classify different microoganisms based on examination of ion patterns from different microoganisms and unique distinguishing protein ions as biomarkers for different strains. The second section, bacteriophages MS2 and T4 were used as materials for identification of their specific protein markers. Protocols including sample preparation, purification, combined in gel digestion and post source decay to which the mass spectrometer brought for analytical specificity were employed. According to the experimental results, classification of different microorganisms based on examination of ion patterns is feasible. Furthermore, the peak at m/z 5255 in the MALDI mass spectra of MS2 analysis and the peak at m/z 8600 of T4 can provide the unique distinguishing signals. The experimental mass spectral peaks were submitted to the database search, and one of these peaks was matched to a tail fiber assembly helper protein. Thus, the method developed and the mass spectra presented in this thesis can be potentially applied to the practical virus analysis.

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The interaction between the matrix and analyte molecules are studied with a high resolution MALDI imaging technique in an orthogonal-injection TOF mass spectrometer. The analyte incorporation and distribution patterns have been clearly demonstrated. Purified protein analytes were found to be homogeneously incorporated in large single crystals of DHB and sinapinic acid matrices, with no evidence for preferred crystal faces. Segregation of some species was observed and appeared to correlate with analyte hydrophobicity, and to a lesser extent analyte mass or mobility. The influence of incident laser parameters on sensitivity in MALDI has been investigated using orthogonal-injection time-of-flight (TOF) instruments. A qualitative comparison was first made between the beam profiles obtained with a N2 laser and a Nd:YAG laser using 2-m long optical fibers. The N2 laser gives better sensitivity, consistent with a more uniform fluence distribution and therefore better coverage of the N2 laser profile. Most of the difference disappears when a 30-m long fiber is used or when the fibers are twisted during irradiation to smooth out the fluence distribution. In more systematic measurements, the total integrated ion yield from a single spot (a measure of sensitivity) was found to increase rapidly with fluence to a maximum, and then saturate or decrease slightly. For a fluence near threshold, the integrated yield has a steep (cubic) dependence on the spot size, but the yield saturates at higher fluence for smaller spots. The area dependence is much weaker (close to linear) for fluence values above saturation, with the result that the highest integrated yields per unit area are obtained with the smallest spot sizes. Finally the detection properties of the MCP detector were studied with a hybrid MCP and CuBe venetian blind converter detector. The measurements show that the detection efficiency of the MCP drops with the increasing of ion mass and the decreasing of the ion energy. The secondary electron emission coefficient of the MCP shows a linear dependence on mass and a power law dependence on velocity (~ 3.2). No clear velocity threshold is observed for secondary electron emission.

The interaction between the matrix and analyte molecules are studied with a high resolution MALDI imaging technique in an orthogonal-injection TOF mass spectrometer. The analyte incorporation and distribution patterns have been clearly demonstrated. Purified protein analytes were found to be homogeneously incorporated in large single crystals of DHB and sinapinic acid matrices, with no evidence for preferred crystal faces. Segregation of some species was observed and appeared to correlate with analyte hydrophobicity, and to a lesser extent analyte mass or mobility. The influence of incident laser parameters on sensitivity in MALDI has been investigated using orthogonal-injection time-of-flight (TOF) instruments. A qualitative comparison was first made between the beam profiles obtained with a N2 laser and a Nd:YAG laser using 2-m long optical fibers. The N2 laser gives better sensitivity, consistent with a more uniform fluence distribution and therefore better coverage of the N2 laser profile. Most of the difference disappears when a 30-m long fiber is used or when the fibers are twisted during irradiation to smooth out the fluence distribution. In more systematic measurements, the total integrated ion yield from a single spot (a measure of sensitivity) was found to increase rapidly with fluence to a maximum, and then saturate or decrease slightly. For a fluence near threshold, the integrated yield has a steep (cubic) dependence on the spot size, but the yield saturates at higher fluence for smaller spots. The area dependence is much weaker (close to linear) for fluence values above saturation, with the result that the highest integrated yields per unit area are obtained with the smallest spot sizes. Finally the detection properties of the MCP detector were studied with a hybrid MCP and CuBe venetian blind converter detector. The measurements show that the detection efficiency of the MCP drops with the increasing of ion mass and the decreasing of the ion energy. The secondary electron emission coefficient of the MCP shows a linear dependence on mass and a power law dependence on velocity (~ 3.2). No clear velocity threshold is observed for secondary electron emission.