The incidence of breast cancer became the most common female cancer, and the fourth cause of female cancer death. In this study, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) have been combined with multivariate statistics to investigate breast cancer tissues and cell lines. Core needle biopsy and fine needle aspiration (FNA) are techniques largely applied in the diagnosis of breast cancer. In this study, we have established an efficient protocol for detecting breast tissue and FNA samples with MALDI-TOF/MS. With the help of statistical analysis software, we can find the lipid-derived ion signals which can be use to distinguish breast cancer tumor tissues from non-tumor parts. This strategy can differentiate normal and tumor tissue, which is potential to apply in clinical diagnoses. The analysis of breast cancer tissue is challenging as the complexity of the tissue sample. Direct tissue analyses by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) allows us to investigate the molecular structure and their distribution while maintaining the integrity of the tissue and avoiding the loss of signals from extraction steps. Combined MALDI-IMS with statistic software, tissues can be analyzed and classified based on their molecular content which is helpful to distinguish tumor regions from non-tumor regions of breast cancer tissue. Our result shows the differences in the distribution and content of lipids between tumor and non-tumor tissue which can be supplements of current pathological analysis in tumor margins. In this study, MALDI-TOF/MS combined with multivariate statistics were used to rapidly differentiate breast cancer cell lines with different estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status. The protocol for efficiently detecting peptides and proteins in breast cancer cells with MALDI-TOF/MS was established, two multivariate statistics including principle component analysis (PCA) and hierarchical clustering analysis were used to process the obtaining MALDI mass spectra of six different breast cancer cell lines and one normal breast cell lines. Based on the difference of the peptide and protein profiles, breast cancer cell lines with same ER and HER-2 status were grouped in nearby region on the PCA score plot. The results of hierarchical cluster analysis also revealed high conformity between breast cancer cell protein profiles and respective hormone receptor types.

Elucidation of the primary structure of peptides and proteins de novo by mass spectrometry (MS) has become possible with the advent of tandem MS methods. The most widely used chemical method due to Edman (Edman & Begg, 1967) has shortcomings with regard to N- terminal blocked peptides, cyclic peptides and posttranslational modifications, for example phosphorylation (Metzger, 1994). However, mass spectrometric sequencing methods are increasingly becoming applicable for a variety of peptides and proteins, including N- and C- termini modified peptides and cyclic peptides (Jegorov et al., 2003; Sabareesh & Balaram, 2006; Sabareesh et al., 2007). Further, conventional and tandem mass spectrometry have proven useful in the detection of post-translational modifications (Hansson et al., 2004; Nair et al., 2006; Mandal et al., 2007). This thesis details mass spectrometric sequencing of acyclic and cyclic peptides, involving tandem MS methods carried out using both electrospray ionization (ESI) ion trap (Esquire 3000 plus, Bruker Daltonics) and matrix assisted laser desorption and ionization time-of-flight/time-of-flight (MALDI TOF/TOF) (Ultraflex TOF/TOF, Bruker Daltonics) instruments. The peptides are either chemically synthesized or isolated from diverse natural sources. Synthetically designed peptides possessing modified N- and C- termini and peptaibols from the soil fungus Trichoderma constitute the acyclic peptides. The cyclic peptides include backbone cyclized depsipeptides from the fungus Isaria and disulfide bonded peptides from the venom of marine cone snails. Chapter 1 gives an account of various concepts of mass spectrometry, tandem mass spectrometry and peptide fragmentation chemistry, providing necessary background information for the following chapters. Chapter 2 describes the fragmentation studies of [M + H]+ and [M + Na]+ adducts of six neutral peptides with blocked N- and C- termini investigated using an electrospray ion trap mass spectrometer. The N- terminus of these synthetically designed peptides is blocked with a tertiarybutyloxycarbonyl (Boc) group and the C- terminus is esterified. These peptides do not possess sidechains that are capable of complexation and hence the backbone amide units are the sole sites of protonation and metallation. The cleavage pattern of protonated adducts is strikingly different from that of sodium adducts. While the loss of the N- terminal blocking group happens quite readily in the case of MS/MS of [M + Na]+, the cleavage of C- terminal methoxy group seems to be a facile process in the case of MS/MS of [M + H]+. Fragmentation of the protonated adducts yields only bn ions, while yn and an type ions are predominantly formed from the fragmentation of sodium adducts. The an ions arising from the fragmentation of [M + Na]+ lack the N-terminal Boc group (termed as an*). MS/MS of [M + Na]+ species also yields bn ions of substantial lower intensities, that lack the N- terminal Boc group (bn*). Comparison of the fragmentation of [M + H]+ with [M + Na]+ of the peptides chosen in this study reveal that the combined use of both protonated and sodium adducts should prove useful in de novo sequencing of peptides that possess modified N- and C- termini, particularly naturally occurring neutral peptides, for example, peptaibols. Chapter 3 describes about the ESI-MS/MS investigation of an HPLC fraction from the soil fungus Trichoderma, which aided in identification of microheterogeneous trichotoxin peptaibols in that fraction. Dramatic differences were noted between the fragmentation spectra of [M + H]+ and [M + Na]+ species. While b-type ions were noted from the former, the latter yielded a-, b-and y- type ions (the same feature was noted in the cases presented in the previous chapter). Inspection of the isotope pattern of b-ions yielded from the dissociation of H+ species, clearly revealed the presence of three microheterogeneous trichotoxin sequences; two isobars (1718 Da), each possessing one Glu residue and another completely neutral peptide (1717 Da). The microheterogeneity is due to Gly ↔ Ala, Iva ↔ Aib and Gln ↔ Glu replacements and exchanges (Iva: DIva: R-Isovaline; Aib: α-aminoisobutyric acid). The MS/MS of [M + Na]+ adduct predominantly yielded product ions from the neutral peptaibol. Further, the fragmentation patterns of H+ and Na+ adducts of two N-acetyl peptide esters were found to be very similar to that of the neutral peptaibol component. The results presented in this chapter establish that under the electrospray ion trap conditions, the fragmentation patterns of the H+ and Na+ adducts of model peptides that possess modified N- (Boc and acetyl) and C- termini are indeed very similar to that of the neutral trichotoxin. Chapter 4 delineates the applicability of liquid chromatography coupled to conventional and tandem electrospray ionization mass spectrometry (LC-ESI-MS, LC-ESI-MS/MS, LC-ESI-MS3) for the screening of novel cyclic hexadepsipeptide metabolites directly from the crude hyphal extract of the fungus Isaria. The fungal strain was grown on a solid medium (potato carrot agar), which yields aerial hyphae growing erect from the basal mycelial colony (Ravindra et al., 2004). A total of ten microheterogeneous components were identified to belong to the isariin class of cyclodepsipeptides from the LC-ESI-MS and LC-ESI-MS/MS analysis of the crude hyphal extract. Out of ten, six are determined to be new and the remaining four are previously reported isariins A-D. The primary structures of isariins A-D were from the fungi Isaria cretacea and Isaria felina (Vining & Taber 1962; Deffieux et al., 1981) and the fungal strain used in this study resembles Isaria felina (Sabareesh et al., 2007). Isariins are backbone cyclized hexadepsipeptides composed of a D-β-hydroxy acid possessing a hydrocarbon sidechain and five α-amino acids; one of the α-amino acids is a D-amino acid (Vining & Taber 1962; Deffieux et al., 1981). The detection of fragment ions due to loss of CO concomitant with the loss of H2O from the protonated precursor ion ([M + H]+) ascertained the cyclic depsipeptide nature of both the known and the new components. The fragmentation behavior of the [M + H]+ of known isariins facilitated sequence determination of the new components. Therefore, the configuration of the amino acids and the β-hydroxy acid of the new components is assumed to be same as that of the reported peptides. The microheterogeneity of the ten sequences is due to changes in the D-β-hydroxy acid (residue 1) and the adjoining α-amino acid (residue 6), whose carbonyl is linked to the hydroxyl function by an ester linkage. The number of methylene units ((-CH2)n) in the hydrocarbon sidechain of the residue 1 differs between 2 and 8 and the variability of the residue 6 is limited to Ala/Val. The ester oxygen atom was chosen as the preferable site of protonation causing ring-opening, based on the observed distribution of the fragment ions. Chapter 5 demonstrates the utility of the LC-ESI-MS and LC-ESI-MS/MS methods in the identification and characterization of six microheterogeneous backbone cyclized hexadepsipeptides, isaridins, directly from the crude hyphal extract of the fungus Isaria. Among the six components, four were found to be novel. The other two peptides, isaridins A and B were identified earlier from this laboratory (Ravindra et al., 2004). The isaridins are characterized by the presence of unusual amino acids such as N-methylated residues, β-methylproline (β-MePro) and hydroxyleucine (HyLeu) (Ravindra et al., 2004). The cyclic nature of both the known and the new peptides were confirmed from the observation of peaks due to loss of CO and H2O from the protonated precursor ion ([M + H]+). However, unlike isariins (Chapter 4), the intensity of the peak corresponding to [M + H - H2O]+ was noted to be of very low intensity, in the case of isaridins. Detection of product ion peak due to [M + H - CO2]+ suggests an additional dissociation pathway involving cleavage at the depsipeptide linkage and is supportive of the cyclic depsipeptide nature (Eckart, 1994). The sequencing of the newly detected components was enabled by understanding the fragmentation mechanism of the known isaridins. The tertiary amide nitrogens of the N-methylated residues were regarded as the preferable sites of protonation leading to ring-opening, as noted from the fragmentation spectra. The microheterogeneity in the sequences was identified using the diagnostic product ions obtained from the protonated precursor of the known isaridins. The microheterogeneity can be attributed to the variations of two residues; Pro ↔ β-MePro and N-MePhe ↔ N-MeLxx (Lxx: Leu, Ile, alloIle). The recently reported ‘isarfelins’ from the fungus Isaria felina (Guo et al., 2005) were reassigned as ‘isaridins’. The reassignment was based on very similar fragmentation profiles observed for the [M + Na]+ adduct of isaridins and isarfelins; further, the fungal strain used in this study resembles Isaria felina (Sabareesh et al., 2007). Chapter 6 presents mass spectrometric sequencing of disulfide bonded peptides from marine cone snails (conopeptides), using the MALDI LIFT MS/MS method. Lo959, a single disulfide bonded octapeptide isolated from Conus loroisii, was identified to belong to the class of contryphans (Sabareesh et al., 2006). Contryphans are small single disulfide bonded conopeptides, whose length is in the range of 7-11 residues and are rich in tryptophan. A significant feature of the contryphans is the presence of conserved DTrp (DW) at the 3rd residue within the disulfide loop (Sabareesh et al., 2006). Lo959 displays an unusual behavior under reverse phase chromatographic conditions, typical of the DW containing contryphans (Jacobsen et al., 1998). It undergoes slow conformational interconversion on the chromatographic time scale exhibiting two distinct peaks. The presence of DW at the 4th position in Lo959 was established by comparing the chromatographic profiles of natural peptide with that of two chemically synthesized peptides, one containing LW (4) and another possessing DW (4). De novo sequencing of the two peptides Ar1446 and Ar1430 from Conus araneosus established that they belonged to M-superfamily of conotoxins, in particular m-2 branch. M-superfamily conotoxins are three-disulfide bonded peptides characterized by the consensus cysteine framework, CC…C…C…CC (Corpuz et al., 2005). Ar1446 and Ar1430 are fourteen residue long peptides, each possessing three disulfide bonds. The peptides have the cysteine scaffold typical of the M-superfamily, as shown above. Specifically, the peptides belong to m-2 branch of M-superfamily, where the fourth and fifth cysteines are separated by two residues (Corpuz et al., 2005). The sequences of the peptides were derived following chemical and enzymatic modifications. The carboxamidomethylation reaction established the presence of three disulfide bonds. Indeed, the sequences were deduced from the MALDI LIFT MS/MS of [M + H]+ of the tryptic peptides. The sequences of the two peptides are almost identical and they differ only at residue 12; hydroxyproline in Ar1446, proline in Ar1430.

L'identification des micro-organismes reposait jusqu'à présent sur l'étude des caractères culturaux et biochimiques de chaque espèce. Depuis quelques années, la spectrométrie de masse de type Matrix Assisted Laser Desorption/Ionization Time Of Flight (MALDI-TOF) s'est développée dans les laboratoires de microbiologie clinique. Cette nouvelle technologie permet de réaliser très rapidement et à moindre coût un diagnostic d'espèce sur des colonies de bactéries ou de champignons isolées sur des milieux de culture solides.Dans un premier temps, nous avons montré que cette technologie permet de réaliser une identification des germes isolés en milieu liquide, comme les flacons d'hémoculture au cours des bactériémies par exemple. Ce dépistage se fait directement à partir du flacon positif, sans attendre l'isolement des colonies sur milieu solide. Ce diagnostic disponible dès le premier jour permet d'adapter l'antibiothérapie au phénotype de résistance habituel de l'espèce.Dans un deuxième temps, nous avons cherché à identifier la nature des biomarqueurs utilisés pour l'identification des espèces bactériennes, en prenant comme exemple la bactérie pathogène Neisseria meningitidis. La comparaison du génome et du protéome des souches entièrement séquencées a permis de mettre en évidence la nature exacte des protéines impliquées dans le diagnostic d'espèce. Par ailleurs, les protéines ribosomales étant majoritaires et pouvant servir d'outil épidémiologique, nous avons constaté que la mise en évidence de leurs variations sur le spectre de masse rend la différenciation de souches au sein d'une même espèce possible, en adaptant la méthode d'analyse. Enfin, nous avons présenté des résultats préliminaires encourageants sur l'exploitation du caractère constant de certaines protéines ribosomales visibles directement sur le spectre de masse, permettant de différencier des espèces très proches, comme Streptococcus pneumoniae et Streptococcus mitis.

Ce manuscrit de thèse présente le développement de méthodes basées sur la spectrométrie de masse consacrées à la recherche d'inhibiteurs d'enzymes en milieux complexes, tels que les extraits de plantes. L’enzyme Tyrosinase a été utilisé comme principale cible biologique du fait de son implication dans les processus d’hyperpigmentation cutanée. De ce fait, la recherche d’inhibiteurs de cette enzyme, présente un grand intérêt pour l'industrie cosmétique. La première partie de ce manuscrit décrit la mise en place de la chromatographie d'affinité frontale (FAC), permettant d’obtenir le classement simultané des inhibiteurs présent dans un mélange complexe en fonction de leurs affinités avec la cible biologique. Deux capillaires hydrophiles de phase monolithiques ont été évalués afin de réduire au maximum les interactions non spécifiques indésirables entre les analytes et le support solide d’immobilisation. De plus, nous avons étudié la faisabilité de l’utilisation de phases à base de silice comme support solide d’immobilisation des enzymes dans le cadre de ces analyses par chromatographie d'affinité frontale. La seconde partie du manuscrit de thèse est consacrée au développement et à l’optimisation de l’approche nommée ENALDI-MS (Enzyme-coupled Nanoparticles-Assisted Laser Desorption/Ionisation Mass Spectrometry) permettant d’accéder à une gamme des faibles masses (m/z 500 Da). Elle est déclinée en une première approche dite par ‘extinction d’ions’ (Ion Fading, IF-ENALDI), basée sur l’identification directe de la liaison des inhibiteurs vis-à-vis de l’enzyme sans pré-traitement de l’échantillon végétal. Une seconde déclinaison de l’ENALDI-MS concerne une approche dite par ‘Ion Hunting’ (IH - ENALDI MS), basée sur une méthode de pré-concentration sélective des inhibiteurs présents dans l'échantillon. / This thesis report presents the development of mass spectrometry-based methods for searching for inhibitors of enzymes in complex mixtures, such as plant extracts. Tyrosinase enzyme was used as the main biological target for the reason of a significant importance of its inhibitors in the cosmetic industry as the skin whitening agents. The first part of this report describes Frontal Affinity Chromatography (FAC), an approach enabling simultaneous ranking the inhibitors within the complex mixture according to their affinities to the biological target. Two hydrophilic capillary-scale polymer-based bioaffinity stationary phases were evaluated in the context of the presence of undesirable nonspecific interactions between the analyte and the solid immobilisation support. In addition, we explored the usability of two types of silica-based particles as a solid support for enzyme immobilisation for FAC. The second part of the thesis manuscript is devoted to Enzyme-coupled Nanoparticle-Assisted Laser Desorption/Ionisation Mass Spectrometry (ENALDI MS) as a low-mass compatible extension of the Intensity ion Fading MALDI MS (IF-MALDI MS) method for high-throughput screening of the inhibitors in the complex mixtures. Two variations of ENALDI MS were evaluated: 'Ion Fading' (IF-ENALDI MS), based on on-the-spot binding of inhibitors by enzyme molecules and 'Ion Hunting' (IH-ENALDI MS), based on selective pre-concentration of inhibitors present in the sample.

Orientador: Marcos Hikari Toyama / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T12:20:31Z (GMT). No. of bitstreams: 1 Oliveira_SimoneCristinaBuzzo_D.pdf: 2257171 bytes, checksum: dafe56777caf52fdf6e83be317bb28f9 (MD5) Previous issue date: 2011 / Resumo: O objetivo deste trabalho foi avaliar o efeito de glicações sobre a PLA2 secretórias isoladas do veneno das serpentes Crotalus durissus collilineatus e Bothrops jararacussu. A técnica de MALDI-TOF foi usado para comprovar a glicação dos carboidratos nas PLA2: a PLA2 de Cdcolli apresentou glicação a 6 moléculas de D-Glicose e de 5 moléculas de D-Lactose, mas aparentemente não houve glicação com nenhuma molécula de D-Galactose; a BthTX-I apresentou glicação com 5 moléculas de D-Glicose, 1 molécula de D-Galactose e 4 moléculas de D-Lactose; a BthTX-II apresentou ligação a 6 moléculas de D-Glicose, a 1 molécula de D-Galactose e a 4 moléculas de D-Lactose; além disso, a dimerização das PLA2 não ocorreu. A glicação da PLA2 de Cdcolli com D-Glicose e D-Lactose aumentou sua atividade enzimática e mudou seu perfil alostérico, da mesma forma, esses carboidratos aumentaram a ação edematogênica e anularam a atividade citotóxica dessa PLA2. No entanto, D-Lactose e D-Glicose diminuíram o efeito miotóxico e modificaram o perfil de agregação plaquetária da PLA2 de Cdcolli nativa, embora apenas a D-Lactose tenha diminuído significantemente a agregação. A glicação de BthTX-I a D-Glicose e D-Galactose diminuiu sua ação edematogênica, e sua glicação aos carboidratos testados diminuiu seu efeito miotóxico. A glicação de BthTX-II a D-Lactose diminuiu sua atividade enzimática, a ação edematogência foi diminuída pela glicação a todos os carboidratos, enquanto a glicação a D-Glicose e D-Galactose diminuíram seu efeito miotóxico. A fluorescência intrínseca apresenta mudanças na estrutura terciária de BthTX-I e BthTX-II após a glicação e o dicroísmo circular sugere modificação na estrutura secundária da PLA2 de Cdcolli. Além disso, a modelação da PLA2 de Cdcolli apresentou várias regiões da proteína livres onde pode estar ocorrendo a glicação. Esse resultados sugerem que a glicação modifica a estrutura da PLA2, modulando de forma diferente a atividade enzimática e o efeito biológico, possivelmente a mudança estrutural ou mesmo os carboidratos ligados a estrutura da PLA2 estão modificando sua afinidade a receptores de membrana. Adicionalmente, os resultados evidenciam que os sítios responsáveis pelas atividades enzimática e biológica estão em lugares diferentes dentro da estrutura da PLA2 / Abstract: The objective of this work was evaluate the effect of glycations in the secretory PLA2 isolated from Crotalus durissus collilineatus (Cdcolli) and Bothrops jararacussu (BthTX-I, catalytic inative K49 and BthTX-II, catalytic active D49) rattlesnake venom. The MALDI-TOF technic was used to test the glycation in the PLA2: Cdcolli showed glycation to 6 molecules of D-Glucose and 5 molecules of D-Lactose, but no glycation to D-Galactose; the BthTX-I showed glycation to 5 molecules of D-Glucose, 1 molecule of D-Galactose and 4 molecules of D-Lactose; the BthTX-II showed glycation to 6 molecules of D-Glucose, 1 molecule of D-Galactose and 4 molecules of D-Lactose. The glycation of PLA2 from Cdcolli to D-Glucose and D-Lactose increased the enzymatic activity and changed its alosteric profile, in the same way, theses carbohydrates increased the edematogenic effect and annuled the citotoxic activity. However, D-Lactose and D-Glucose decreased the miotoxic effect and changed the platelet aggregation profile of native PLA2, although only D-Lactose had decreased the aggregation. The glycation of BthTX-I to D-Glicose and D-Galactose decreased the edematogenic effect and its glycation to all carbohydrates decreased the miotoxic effect. The glycation of BthTX-II to D-Lactose decreased its enzymatic activity; the edematogenic effect was decreased by the glycation to all the carbohydrates, while the glycation to D-Glucose and D-Galactose decreased the miotoxic effect. The intrinsec fluorescence shows changes in the terciary structure of BthTX-I and BthTX-II after glycation and the circular dichroism suggests modification in the secundary structure of glycated PLA2 from Cdcolli. Besides, the structure modelation shows many free areas in the PLA2 from Cdcolli, where the glycation can be happening. Theses results suggest that the glycation modifies the structure of PLA2, modulating in a different way the enzymatic and the biologic activity; probably the structure changes or even the linked carbohydrates to the PLA2 structure are modifying the affinity for membrane receptors. Additionally, the results show that the enzymatic and biological sites are in different areas in the PLA2 structure. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

O gênero Trichosporon é composto por leveduras artrosporadas do Filo Basidiomycota e é conhecido agente de infecção fúngica invasiva (IFI) em pacientes imunodeprimidos ou com outros fatores de risco. Em pacientes onco-hematológicos é a principal levedura responsável por IFI depois do gênero Candida. Entre as espécies responsáveis por infecções no homem encontram-se: T. asahii, T. inkin, T. mucoides, T. dermatis, T. jirovecii, T. ovoides, T. cutaneum, T. montevideense, T. domesticum, T. asteroides, T. coremiiforme, T. faecale, T. dohaense, T. lactis, T. japonicum. A tecnologia de identificação de fungos por espectrometria de massa (SM) MALDI-TOF ainda carece de padronização para identificação de fungos do gênero Trichosporon, mas a literatura mostra resultados encorajadores. O objetivo deste estudo é padronizar a técnica de espectrometria de massa MALDI-TOF para a identificação das espécies do gênero Trichosporon de importância médica. O estudo foi realizado em cooperação entre a Divisão de Laboratório Central do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (DLC, HC-FMUSP), Instituto de Medicina Tropical da USP (IMT-USP), Instituto Adolfo Lutz (IAL) e Laboratoire de Parasitologie-Mycologie do Hospital Saint Antoine de Paris, vinculado ao grupo de pesquisa INSERM/UPMC UMR S945 \"Immunité et Infection\" Faculté de Medecine et Université Pierre et Marie Curie de Paris. Noventa e três cepas/isolados foram analisado(a)s, sendo dezenove cepas de referência adquiridas junto à coleção holandesa Centraalbureau Schimmelcultures (CBS), 19 isolados do HC-FMUSP e IAL, e 55 isolados de diferentes hospitais franceses. A identificação molecular foi realizada através do sequenciamento da região IGS1 do rDNA e foi considerada como método de referência. O protocolo de extração de proteínas foi estabelecido através da comparação do desempenho de três metodologias (Bruker®, Cassagne et al., Sendid et al.). Os espectros de massa foram obtidos no laboratório de bacteriologia do Hospital Saint Antoine de Paris através do aparelho Microflex LT®. A interpretação dos resultados qualitativos e quantitativos (logscore) foi realizada através do Software Biotyper 3.0®. O desempenho de identificação do banco de espectros de referência Biotyper 3.0® foi comparado a outros cinco bancos criados a partir de espectros de referência (ERs) derivados de 18 cepas de referência CBS, sete isolados clínicos e 11 ERs do banco Biotyper 3.0. O protocolo de extração de proteínas descrito por Sendid et al. foi escolhido como protocolo de referência pois os espectros produzidos tiveram logscore superiores àqueles obtidos através do método do fabricante. O banco de ERs Biotyper 3.0® apresentou 32,3% de identificações corretas das espécies, sendo que o banco de ERs in house (número 5, constituído cepas CBS e isolados clínicos) apresentou 98,5% de identificações de espécies. Espectros de referência do banco de dados Biotyper 3.0® foram submetidos à identificação com a utilização dos ERs criados a partir de cepas CBS e isolados clínicos e foram evidenciados com erros de identificação: T. mucoides (2), T. ovoides (1) e T. cutaneum (2). Após padronização do protocolo de extração e criação de banco de ERs com cepas CBS e isolados clínicos caracterizados pelo sequenciamento da região IGS, a SM por MALDI-TOF apresentou-se como potente uma ferramenta para a identificação de fungos do gênero Trichosporon. O banco de ERs Biotyper 3.0® apresentou um fraco desempenho, relacionado a ERs que foram criados a partir de cepas mal identificadas / Trichosporon spp. are arthrospored yeasts from the Filum Basidiomycota that are known to produce invasive fungal infection (IFI) in patients with immunosupression or other risk factors. After Candida, Trichosporon is the second genus of yeasts responsible for IFI in patients with onco-hematological diseases. The most important species related to human infection are: T. asahii, T. inkin, T. mucoides, T. dermatis, T. jirovecii, T. ovoides, T. cutaneum, T. montevideense, T. domesticum, T. asteroides, T. coremiiforme, T. faecale, T. dohaense, T. lactis, T. japonicum. The technology of mass spectrometry (MS) for identification of Trichosporon species has not yet been standardized. However, preliminary promising results can be found in the literature. The objective of this study is to analyse and validate MS MALDI-TOF for the identification of Trichosporon species of medical relevance. This was a multicentric study with collaboration from the Central Laboratory Section from Clinics Hospital of the Medical School from the University of São Paulo (DLC-HCFMUSP), Tropical Medicine Institute from the University of São Paulo (IMT-USP), Instituto Adolfo Lutz (IAL) and Laboratoire de Parasitologie-Mycologie from the Hospital Saint Antoine of Paris and INSERM/UPMC UMR S945 \"Immunité et Infection\", Faculté de Medecine et Université Pierre et Marie Curie of Paris. Ninety three strains/isolates belonging to sixteen Trichosporon species were analysed. Nineteen were purchased from Centraalbureau Schimmelcultures (CBS) yeast collection, 19 belonged to HC-FMUSP and IAL collections, 55 belonged to different French collections. The reference identification method was the IGS1 rDNA sequencing. A protein extraction protocol was first established after comparing the performance of three different methodologies (Bruker(TM), Cassagne et al., Sendid et al.). The mass spectra were obtained through a Microflex LT(TM) mass spectrometer located at the bacteriology laboratory from Saint Antoine Hospital, Paris. Mass spectra, qualitative and quantitative results were produced through the software Biotyper 3.0(TM). The performance of the original main spectrum (MSP) library was compared to other 5 in house libraries built with the combination of MSPs derived from CBS strains (18), clinical strains (7) or (Bruker Daltonics/BD, Germany/USA) (11). The extraction protocol described by Sendid et al. showed better performance when compared to the manufacturer\'s one and was chosen for the subsequent extractions. Among the 6 different reference spectra databases tested, a specific one composed of 18 reference strains plus 7 clinical isolates (database 5) allowed the correct identification of 66 amongst 67 clinical isolates (98,5%). Biotyper 3.0 library produced only 32,3% of correct identifications. Biotyper\'s MSPs were submitted to cross-identification with MSPs derived from CBS strains and clinical isolates and misidentified original MSPs were identified: T. mucoides (2), T. ovoides (1) e T. cutaneum (2). While until now less widely applied to basidiomycetous fungi, MALDI-TOF appears to be a valuable tool for identifying clinical Trichosporon isolates at the species level. The MSP library Biotyper 3.0 showed a poorer performance which was due to misidentified strains utilized as reference for the MSPs

Today we are collecting a large amount of tissue samples to store for future studies of different health conditions, in hopes that the focus in health care will shift from treatments to early detection and prevention, by the use of biomarkers. To make sure that the storing of tissue is done in a reliable way, where the molecular profile of the samples are preserved, we first need to characterise how these changes occur. In this thesis, data from mice brains were collected using MALDI imaging mass spectrometry (IMS) and an analysis pipeline for robust MALDI IMS data handling and evaluation was implemented. The finished pipeline contains two reduction algorithms, catching images with interesting intensity features, while taking the spatial information into account, along with a robust similarity measurement, for measuring the degree of co-localisation. It also includes a clustering algorithm built upon the similarity measurement and an amino acid mass comparer, iteratively generating combinations of amino acids for further mass comparisons with mass differences between cluster members. Availability: The source code is available at https://github.com/stephanieherman/thesis

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the field of bacterial diagnostics and is also used for routine analysis in smaller clinical laboratories. For identification of moulds, pre-analytical steps are more complicated and time consuming than for bacteria, and the choice of reference library has a big impact on the utility of MALDI-TOF MS. The aim of this study was to investigate if MALDI-TOF MS is applicable for identification of moulds belonging to the genus Aspergillus at the hospital laboratory in Gävle. Therefore, strains belonging to the genus Aspergillus and Penicillium, were analysed with MALDI-TOF MS after 2, 4 and 7 days of incubation. Two different extraction protocols were used and compared. Mass spectra were compared to reference spectra in two different databases: MSI-2 and RUO Compass library/BDAL (Bruker). Of the strains included, 97 % were correctly identified to species complex level with MSI-2. Only 25 % were identified to species level with RUO Compass library/BDAL (Bruker). However, totally 56 % were correctly identified to species complex level if a lower score value limit than recommended for identification, were applied. Significantly raised score values were observed with one of the protein extraction protocols used. Although, in most cases, the strains were considered identified to species complex level with either method. This pilot study conveys the feasibility of MALDI-TOF MS for identification of Aspergillus species in a clinical laboratory. While there are still issues to address, applying MALDI-TOF MS has the potential to allow for quicker and more precise identification, also in this specific clinical setting.

As permanent tattoos are becoming more popular and common, an increased number of allergic reactions to tattoos is reported. The purpose of this project was to analyze tattoo inks for hazardous substances, and whether they comply to current Swedish and European legislative requirements. The tattoo inks were qualitatively analyzed for pigments, and quantitatively analyzed for metals. A total of 73 tattoo inks were collected from various sources such as a tattoo ink supplier, online retailers, and provided directly from tattoo artists. The labels of each tattoo ink bottle were inspected to investigate their compliance with the Council of Europe and the Swedish Medical Products Agency. Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF-MS) was used to qualitatively analyze 20 selected tattoo inks for different pigments. Inductively coupled plasma mass spectrometry (ICP-MS) was used to quantitatively analyze trace metals in 70 of the samples. A large majority (90%) of the tested samples violated the requirements and criteria in the European resolution ResAP 2008(1), such as information on name and address of the manufacturer, minimum date of durability, sterility, batch number, and storage. Patch and allergy testing were incorrectly recommended for many samples in a way that is not accepted by dermatologists. In a worst-case scenario, this testing could be a sensitizing step. Also, it can not prevent future allergic reactions from occurring or provide any juridical insurance. Only one brand, World Famous, fulfilled the requirements for labeling for six of the seven samples (one sample failed due to a faulty declared pigment). The brands Tang Dragon and Dynamic did not fulfill any of the requirements listed in ResAP 2008(1). The list of ingredients was incorrect for all samples from Tang Dragon (bought prior to 2019 online). Also, six of the other 50 samples from different brands (World Famous, Intenze, Fusion Tattoo Ink, Eternal Ink, Solid Ink) declared at least one pigment incorrectly in their ingredients list. 25% of the declared and theoretically detectable pigments were detected by means of MALDI-ToF-MS, whereas the other pigments were either absent or below the limit of detection. Future analyses should include an MS/MS analysis. Polyethylene glycol (PEG) was identified qualatively in 15 of the 20 samples analyzed with MALDI-ToF-MS but was not listed in any of the ingredients lists. ICP-QQQ-MS is a very sensitive technique and could both detect and verify the presence of all metal-containing pigments, as well as the level of impurities. Copper was clearly more present in green and blue colors, regardless of the brand. The metal content was evidently dependent on the brand for arsenic, aluminum, bismuth, chromium, nickel, zinc, and strontium. Elevated levels of barium and strontium (partially very high levels: up to 727 mg/kg barium and up to 8.06 g/kg strontium) were found in several samples. High amounts of aluminum (4 to 11,0 g/kg) and titanium (as judged from white precipitates and ingredients lists) were present in most samples. Nickel (0.1 to 41 mg/kg) and chromium (0.1 to 139 mg/kg) were also present in the samples. Some other impurities were also present (arsenic – 3.8 mg/kg, mercury – 1.6 mg/kg, and lead – 5.4 mg/kg for one sample, respectively). Known sensitizing pigments were declared and partially confirmed by MALDI-ToF-MS in 17 of 53 samples of the brands Radiant Colour, Eternal Ink, Fusion Tattoo Ink, and Kuro Sumi. Four samples (from Intenze, Eternal Ink, and Kuro Sumi) also declared pigments listed as non-suitable substance according to the European Commission regulation on cosmetic products from 2009.

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