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MALDI imaging allows the creation of a molecular image of a tissue slice. This image is reconstructed from the ion abundances in spectra that are obtained while rastering the laser over the tissue. These images can then be correlated with tissue histology to detect potential biomarkers of, for example, aberrant cell types. MALDI is known to have problems with ion suppression, making it difficult to correlate measured ion abundance with concentration. It would be advantageous to have a method that can provide more accurate protein concentration measurements, particularly for screening applications or for precise comparisons between samples. My hypothesis was that a method based on multiple reaction monitoring (MRM) with isotopically-labelled internal standards can be developed which would allow the accurate quantitation of proteins in MALDI Imaging. This study reports on the development of this novel MALDI Imaging method for the localization and accurate quantitation of proteins in tissues. This method involves optimization of in-situ tryptic digestion, followed by reproducible and uniform deposition of an isotopically-labelled standard peptide from a target protein onto the tissue, using an aerosol-generating device. Data is acquired by MALDI-MRM-MS and accurate peptide quantitation is determined from the ratio of MRM transitions for the endogenous unlabelled proteolytic peptides to the corresponding transitions from the applied isotopically-labelled standard peptides. In a parallel experiment, the quantity of the labelled peptide applied to the tissue was determined using a standard curve generated from MALDI-TOF-MS data. This external calibration curve was then used to extrapolate the quantity of endogenous peptide in a given area. All standard curves generated by this method had coefficients of determination greater than 0.97. These proof-of-concept experiments using MALDI MRM-based imaging show the feasibility of obtaining precise and accurate quantitation of tissue protein concentrations over two orders of magnitude, while maintaining the spatial localization information for the proteins. / Graduate

Orientador: Fabiana Ferreira de Souza / Resumo: O ciclo estral da vaca é composto por 2-3 ondas de crescimento folicular, no qual vários folículos são recrutados e iniciam um novo crescimento. Durante o período denominado desvio folicular, um folículo se torna dominante e os outros entram em atresia. Este processo envolve um mecanismo ainda não completamente compreendido, incluindo proteínas específico, como já estabelecido pela expressão gênica. O objetivo do presente estudo foi caracterizar as proteínas do fluído folicular a fim de identificar macromoléculas relacionadas ao desenvolvimento dos folículos de vacas zebuínas nã-gestantes. Foram colhidos os ovários de 25 vacas mestiças não-gestantes em um abatedouro. A presença do corpo lúteo foi anotada para cada ovário. O líquido folicular foi colhido utilizando-se a imersão do ovário em meio líquido e ultrassonografia. De acordo com a mensuração do diâmetro folicular, foram formados 3grupos, folículos pequenos (≤6,5mm, n=25), médios (>6,5mm a ≤9mm, n=9) e grandes (>9,0mm, n=11). Após 2 centrifugações (600xg/10 minutos e 15.000xg/30 minutos, 4ºC) o sobrenadante foi separado e utilizado para determinação da concentração de proteína total (método de Bradford). A eletroforese foi conduzida sob condições desnaturantes e redutoras, em gel de separação de poliacrilamidaà 12%. A concentração de progesterona e estradiol do líquido folicular foi determinada a fim de identificar os folículos saudáveis. As proteínas diferenciais identificadas pela eletroforese foram... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Acinetobacter baumannii est une bactérie responsable d’infections communautaires, en particulier dans les régions tropicales et chez des blessés lors de conflits armés. Or, les réservoirs extra-hospitaliers de la bactérie sont peu connus. L’objectif de ce travail était d’étudier ces réservoirs dans une zone tropicale, l’île de la Réunion, en étudiant à la fois le réservoir animal (dépistage), environnemental et humain. En parallèle, des études ont été conduites en zone tempérée (France métropolitaine), avec le dépistage des animaux domestiques. Dans un premier temps, l’identification des espèces du complexe Acinetobacter calcoaceticus-Acinetobacter baumannii par spectrométrie de masse a été perfectionnée sur l’automate VitekÒ MS. La deuxième partie du travail a permis de mettre en évidence une prévalence élevée de portage de la bactérie chez les animaux domestiques réunionnais (6,5 et 8,5%). Une prévalence inférieure a été décelée chez les animaux domestiques de métropole (2,7%). Dans tous les réservoirs étudiés, une grande diversité des souches a été retrouvée lors de l’analyse par Multi-Locus Sequence Typing, avec la mise en évidence de nouveauxSequence Types. Enfin, 2 souches productrices d’OXA-23 et OXA-24 ont pu être isolées respectivement en portage chez un patient et un animal d’élevage de l’île de la Réunion, et 2 souches productrices d’OXA-23 ont été isolées chez des animaux de compagnie de métropole. Ce travail a permis des observations intéressantes sur l’épidémiologie communautaire d’ A. baumannii. D’autres travaux restent à prévoir pour mieux comprendre les relations entre les réservoirs extra-hospitaliers, mais aussi avec le réservoir hospitalier. / Acinetobacter baumannii is a bacterium responsible for community-acquired infections, especially in tropical areas and among soldiers during warfare. However, community reservoirs of this bacterium are not very well known. The aim of this work was to study these reservoirs in a topical area, Reunion Island, by analyzing at the same time the animal (carriage), the environmental, and the human reservoirs. Studies were also performed in a temperate zone (metropolitan France), by analyzing carriage of companion animals. First, identification of species belonging to the Acinetobacter calcoaceticus - Acinetobacter baumannii complex by mass spectrometry has been improved on the VitekÒ MS automated system. In asecond part of the study, a high prevalence of A. baumannii carriage has been observed in domesticated animals of La Reunion Island (6,5 and 8,5%). A lower prevalence has been detected in metropolitan companion animals (2,7%). Within allthe reservoirs studied, a great diversity of strains isolated has been shown by Multi-Locus Sequence Typing analysis, with the dicovery of new Sequence Types. At last, 2 strains producing OXA-23 and OXA-24 have been isolated respectively in carriage in a patient and in a livestock animal of La Reunion Island, and 2 strains producing OXA-23 have been isolated in metropolitan companion animals. This work has permitted to do some interesting observations on the community epidemiology of A. baumannii. Other studies are needed to better understandrelationships between the different community reservoirs, and also with the hospital reservoir.

New methods for drugs analysis, disease diagnosis, and protein analysis by using modern mass spectrometry are developed in this thesis. In drugs analysis, we develop a rapid assessment of molecular similarity between an extremely complex innovator product and a candidate biosimilar by mass spectrometry. Protease digestion combined with Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry were successfully used to peptides mapping and de novo peptides sequencing. Overall ion signals obtained by MALDI-TOF/MS were processed by two multivariate statistics including principle component analysis (PCA) and hierarchical clustering analysis. Based on the variances of the peptide profile, innovator product and normally synthesized biosimilar were grouped on the PCA score plot, while impure biosimilar or abnormally synthesized biosimilar were distinctly separated. The results of hierarchical cluster analysis also revealed high conformity between innovator product and normally synthesized biosimilar. Abnormally synthesized products, as a set of quality controller, could be discriminated from innovator product favorably. Another quality control strategy developed in this study is building classification model, batches of product is successfully evaluated. Furthermore, the similarity of molecular weight distribution between these complex drugs is determined. Another target of drug analysis is to develop a vital analytical method to directly detect the peptides synthesized on the resin. Except an organic solvent is transformed to disperse the resin-peptides samples, no other sample pretreatments are required before the MS detection. When using conventional destructive analytical methods to characterize masses of compounds on the solid supports, acid hydrolysis or acid cleavage of the peptide molecules depart from insoluble resin is required. In consequence, side-reactions such as de-blocked or de-protected cause additional fragments in the system, and determination of the intermediates or products are confusing and difficult. Unlike these acid release methods, the molecular weight information of the intact peptide molecules can be obtained in our direct analyses system, and sample consumption is also great reduced. Moreover, our strategy performed the analysis in the ambient environment is more straightforward for real-time monitor reaction and quality control than those techniques in high vacuum system. This direct non-destructive on-line monitoring method would allow following step by step peptide solid-phase synthesis be well quality controlled. In the disease diagnose part, MALDI-TOF mass spectrometry combined with statistics were used to perform semi-quantitative albuminuria diagnoses. Based on the fact that the contributions of singly, doubly, triply, and quadruply charged albumin ions from the samples were inflected according to the concentration change. The severity of albuminuria of patients can be estimated by 2D peaks distribution (peaks ranking by p-value) or supervised principal component analysis (PCA). In protein analysis study, we use liquid electrospray laser desorption ionization mass spectrometry (liquid-ELDI/MS) to directly characterized the proteins stored in different solutions containing acids, bases, buffers, organic solvents, or detergents without extra sample pretreatment. A drop of the sample solution was applied on a stainless steel plate., and then the surface of the sample drop was irradiated with a pulsed laser. The laser energy absorbed by the metal plate and surrounding solvent led to the desorption of protein molecules or the formation of fine droplets containing protein molecules. The desorbed protein species were then post-ionized within an electrospray plume to generate the ESI-like protein ions. Since no pH or composition adjustment of the sample solution is needed, this technique is useful for rapid and high throughput screening of the proteins in a solution to check their integrality after storage or prior to further biochemical treatment. In addition,, native and denatured conformation of the proteins can be distinctly examined by using acid-free and organic solvent-reduced ESI solutions in liquid-ELDI.

In this study, we collected 18 plasma samples from NPC patients, and 10 plasma samples from healthy person for plasma proteomic analysis. We classified these samples into 3 groups: treatment, pretreatment and recurrent. Two-dimensional electrophoresis (2-DE) and MALDI-TOF were performed followed by comparative and statistic analysis. In conclusions, we totally identified 30 proteins in Nasopharyngeal carcinoma (NPC) plasma, and found 10 proteins with expression level down regulated (p<0.001). These proteins were characterized to be Serotransferrin, Vitamin D-binding protein (VDB), alpha-1 antitrypsin (AAT), Haptoglobin, Apolipoprotein B fragment, Syntaxin-7, Apolipoprotein, A-I, PRO1779, Transthyrin, MDN1 protein, respectively. Consequently, we established a protocol to remove high abundant proteins (e.g., albumin, immunoglobin etc.) in plasma. We are especially interested in ATT and VDB. Western blotting assay was performed to confirm ATT and VDB expression. Furthermore, the quantity of ATT and VDB were measured by ELISA to obtain the threshold value of these proteins. Finally, we want to realize the relationship between these down regulation proteins and clinical parameters in NPC malignancy and tumor progression. Since there are few protein expression research of NPC in clinical studies, our works will provide insights in NPC studies for tumor progression with potential to elevate treatment efficiency.

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The motivation of this dissertation is to provide insight towards primary ionization mechanisms within MALDI mass spectrometry. Albeit MALDI-MS is an extensively used analytical technique, the mechanism in which primary ions are created is still under scrutiny. Two current models of primary ionization exist which claim to elucidate the ion formation mechanisms within MALDI. In this work, excited state dynamics of MALDI matrices are shown to play an important role in the ionization mechanism. Upon inspection of the thermodynamic properties of commonly used MALDI matrices, no correlation was observed when plotted against their respective analyte ion yields. However, the excited state singlet lifetimes of these matrices seem to correlate well with their respective analyte ion yields. In the broadest sense, this correlation further supports the fact that photophysical properties of the matrix should be included in current UV-MALDI models. Investigation of a claim which stated singlet energy pooling reactions were absent in the MALDI matrix 2,4,6-trihydroxyacetophenone (THAP) resulted in the discovery of a new energy pooling mechanisms. Characteristic of aromatic ketones such as THAP, intersystem crossing is an efficient process in solution, which gives way to fluorescence in the solid state. Triplet pooling mechanisms from two neighboring THAP molecules are proposed and appear to be dependent on the preparation solvent used. These triplet pooling reactions are thought to play an important role in the primary ion formation mechanism within MALDI. To further investigate the theory of triplet species playing a vital role in MALDI ionization, the internal heavy-atom effect was employed to determine the effect of the triplet species. MALDI mass spectra and excited state decays of these heavy-atom substituted matrices were collected to demonstrate the relationship between triplet species and analyte ionization efficiency. Gas-phase thermodynamics and absorption at 337 nm were also examined to determine if these properties affected the analyte ion signal observed in the MALDI mass spectrum. Using the information collected from the previous study, an advanced MALDI matrix is synthesized. Addition of covalently bound iodine to the gold standard matrix, α-cyano-hydroxycinnamic acid, should drastically improve the performance of the non-substituted matrix due to the increase in triplet species present for pooling reactions. Sample preparation methods in MALDI are examined as are the effects of crystal morphology on the overall signal observed in the mass spectrum. Exciton hopping and pooling rates are highly dependent on intermolecular interactions, so it is expected that crystal packing will affect MALDI. As noted for THAP, preparation solvent plays a significant role in not only crystal morphology, but also the excited state dynamics for all matrices studied.

The work described in this dissertation is divided into three sections. In the first section three surface modifications are used to produce MALDI targets having reduced surface-protein binding affinity with a goal of increasing peptide/protein MALDI ion signals and lowering the limits of detection (LODs) for proteins and peptides. The second section discusses a bioselective MALDI target, produced via radio frequency (rf) plasma deposited ethylenediamine (EDA), for on-target separation of complex protein mixtures. The third section develops a new approach for characterization of rf plasma-deposited bulk polymers by using MALDI MS. Previous studies in our group have shown that the analyte signal in a MALDI MS experiment is strongly influenced by the binding interactions between the target surface and the analyte. Specifically, the analyte signal increases with decreasing surface-analyte binding affinity, which has been attributed to more unbound analyte being available for incorporation within the MALDI matrix. In the presented studies MALDI targets are modified with polyethylene glycol (PEG)-like structures via chemical grafting of PEG onto polyurethane (PU) film and rf plasma polymerization of ethylene oxide vinyl ether (EO2) and tetraglyme. It is shown that there are enhancements in the protein MALDI ion signals on these modified targets and that the LOD for target proteins is decreased by a factor of 2-10 in comparison with the conventional stainless steel MALDI target. On-probe affinity capture (OPAC) MALDI MS, developed in our group, has shown that functional group modified MALDI targets can be used to rapidly and selectively isolate target analytes from complex samples. For applications involving analysis of complex peptide/protein mixtures, fractionation of the mixture on the basis of component pI can reduce MALDI ion suppression effects leading to efficient ionization of larger numbers of mixture components. In the present studies a MALDI target is modified by rf plasma deposition of polymerized EDA to yield an OPAC target suitable for capture of proteins with low pI (expected to be negatively charged at neutral pH). In subsequent MALDI MS analyses of both control and biological mixtures after fractionation on the OPAC target it is observed that a significant number of additional peptide/protein ion signals are detected. The results of these studies, along with studies of the effects of the density of the primary amine functionality on the bio-selective MALDI ion signals, are presented. The complex nature of the polymer films resulting from plasma polymerization makes it very difficult to characterize their molecular structures. The presented study is the first to use MALDI MS for characterization of rf plasma-deposited bulk polymers and for investigation of the rf plasma polymerization process. It is shown that the mass spectra of the soluble fraction of allyl alcohol, EO2 and ethylene glycol butyl vinyl ether -plasma polymers contain clear polymer series. Furthermore, it is found that the peaks of the EO2-plasma polymer series shift to higher molecular weight distribution with decreasing plasma duty cycle. In contrast to predictions based on conventional radical polymerization, the mass spectra of all three plasma polymers exhibit the same repeat unit of 44 Da, for which the most likely structure would be -(CH2CH2O)-.

This thesis describes the use of mass spectrometric methods based upon electrospray ionization, ESI, and (matrix-assisted) laser desorption/ionization, (MA)LDI, for the quantitative analysis of small biomolecules. Structure analysis when required, was obtained through tandem mass spectrometry (MS/MS). The Girard type reagent, 4-hydrazino-4-oxobutyl tris(2,4,6 trimethoxy)phenyl phosphonium bromide, in combination with the solid phase derivatization technique, is used to selectively prepare a pre-ionized malondialdehyde derivative to be analyzed by MALDI or LDI. The in situ derivatization and isolation minimize interferences from other components in biological samples. The combination of pre-ionization and aromatic functionalities allows for laser induced ionization without the need of matrix. The combination of these techniques provides an avenue for development of automation to produce a high throughput method of analysis. Chapter 3 involves the study of the complexation of diols to the oxovanadium ion. The oxovanadium (IV) complex of ethylene glycol is used as a reference to study the complexation of other diols and amines with the vanadyl ion. The ES spectra of various diols studied produce intense signals for the mixed and the analyte complexes, indicating effective complexation of the analytes with the vanadyl ion. Oxovanadium (IV) is observed to be more selective for complexation to diols than amines. This eliminates the possibility of interference from N-containing ligands to the detection of diols by the reference complex. The electrospray spectrum is used for quantitation and the tandem mass spectrometry spectrum for structure confirmation. The MS/MS spectrum also assists the identification of the diols by the structural differences within their isomers. The equilibrium constant of a set of diols was determined and its calibration curve were constructed. This study produces an alternative method to detect and quantify diols in aqueous solutions and blood samples. / Thesis / Master of Science (MSc)