Accumulated evidence indicates oxidative stress plays important roles in disease and aging. Under oxidative stress, lipid peroxidation (LPO) leads to reactive carbonyl species (RCS) that can modify a wide range of biomolecules including protein, DNA and carbohydrate. In this dissertation, we investigate the modification of two model proteins, human serum albumin (HSA) and aconitase (ACO), by the LPO-relevant a, b-unsaturated aldehydes, acrolein (ACR) and 4-hydroxy-2-nonenal (HNE). The investigation is focused on the characterization and quantification ACR and HNE addition to the model proteins. A correlation between HNE modification and ACO activity is also determined. These results provide insights into the impact of oxidative stress at the molecular level and are relevant to aging and disease states. We finally investigate protein carbonylation in ischemic mouse heart mitochondria, and develop a quantitative method for detecting carbonylated protein in this system. The research is based on liquid chromatography/mass spectrometry (LC/MS), Western Blots, and enzymatic assay.

Calcium (Ca2+)/calmodulin-dependent kinase 2 (CaMK-II) is one member of a family of Ca2+/calmodulin-dependent protein kinases that responds to intracellular Ca2+ signals (Hudmon, A. and H. Schulman (2002)). CaMK-II is a multifunctional regulator of transcription, cell cycle progression, cell motility and neuronal development. (Wang, C., et al. (2008), Easley, C. A. IV, et al. (2008), Osterhoff, M., et al. (2003), Faison, M. O., et al. (2002)). Recently, CaMK-II has been shown to be important in the early development of vertebrates. In developing zebrafish, disruption of CaMK-II expression has been shown to induce phenotypes similar to those documented in several human diseases. The identification of the tissue-specific binding partners and substrates of CaMK-II which are responsible for specific developmental fates remains a key step in understanding this important protein kinase family. In this thesis research, specific “substrate-trapping” mutants of CaMK-II were designed, introduced into a variety of rodent and human cell lines in culture and used in conjunction with tandem mass spectrometry to identify binding partners, such as β-actin, tropomodulin-3 and Fli-I as well as novel, putative substrates, such as the tumor suppressor protein 53 (p53). This approach was subsequently applied to zebrafish embryos where an overlapping subset of CaMK-II binding proteins to those found in mammalian cell culture were identified. This project represents one of the first studies to identify binding proteins in zebrafish embryos using epitope tagging and mass spectrometry. This research has also established a technical framework for the use of mass spectrometry to characterize the developmental proteome of whole zebrafish embryos or specific zebrafish tissues at any developmental time point.

In this thesis, the performance of a miniaturised field asymmetric waveform ion mobility spectrometry (FAIMS) device hyphenated with time-of-flight mass spectrometry is studied and evaluated for analysis of a variety of compounds in different sample matrices. FAIMS is a selective spectrometer which is highly orthogonal to mass spectrometry and has the potential for enhancing sensitivity and improve selectivity of rapid analyses. In Chapter 2, the performance of the miniaturised FAIMS device is tested for stability and transmission under a wide range of ion source conditions. An investigation of three different systems, including pairs of isobaric, isomeric and near-mass ions shows that miniaturised FAIMS has the ability to distinguish between analytes that are challenging to separate by mass spectrometry. Chapter 3 explores the effect of changing the composition of the carrier gas by observing the effect of adding gas modifiers on the FAIMS spectra of small molecules, peptides and proteins. Chapter 4 investigates the advantages of combining a fast FAIMS separation with mass spectrometry in the analysis of nitrogen-containing pharmaceutical impurities, where FAIMS is found to offer additional selectivity. In Chapter 5, the development of a UHPLC-FAIMS-MS method for the quantitative determination of a drug metabolite in urine is reported. UHPLC-FAIMS-MS shows improvements in signal-to noise and linear dynamic range as well as a reduction in chemical noise, demonstrating the potential of combining FAIMS with mass spectrometry.

Eleven tested anions were able to form adducts with neutral oligosaccharides at low cone voltage in negative ion mode electrospray mass spectrometry. Among them, fluoride and acetate have the abilities to significantly enhance the absolute abundance of [M-H]- for neutral oliogosaccharides. The chloride adduct has the best stability among all the adduct species investigated. For the above three anions, CID of adduct species may be used for structural determination of neutral oligosaccharides. In the presence of F- and Ac-, simultaneous detection of acidic oligosaccharides and neutral oligosaccharides was achieved. The ratio of Cl- : non-Cl-containing product ions obtained in CID spectra of chloride adducts of disaccharides was used to differentiate anomeric configurations of disaccharides. Density functional theory (DFT) was employed to evaluate the optimized structures of chloride adducts of disaccharides. The formation and decomposition of chloride adducts with oligosaccharides of different acidities were investigated in MALDI mass spectrometry.

The anti-cancer agent 4, 4-dihydroxybenzophenone-2, 4- dinitrophenylhydrazone (A007) forms complexes with pdelocalized lymphangitic dyes that allow its penetration through the skin effectively delivering it to a meta-stable type cancerous site. Previous in vitro studies, combined with gas phase mass spectrometry studies, have shown that a stronger binding affinity equates to a greater efficacy of the drug. For the determination of drug:dye complex binding strength coefficients in solution, two methods have been developed by affinity capillary electrophoresis (ACE), and cation exchange liquid chromatography (CELC). The methods demonstrated that A007 non-covalent binding strength was greatest for methylene green, followed by methylene blue, and lastly toluidine blue. Bond dissociation energies and apparent reaction enthalpies for the fragmentation pathways of lithiated acylglycerols were experimentally determined by collision activation in a triple quadrupole mass spectrometer. A developed novel derived effective path length approach for predicting bond dissociation energies (BDE) for electrostatic complex's alkali metal adducts (Li+), and halide adducts (Cl-) of acylglycerols was applied to the major fragmentation product ions of a lithiated mono-acylglycerol, a 1, 2-diacylglycerol, and a 1, 3-diacylglycerol, to predict the covalent bond dissociation energies involved in fragmentation pathways. The model's calculated apparent reaction enthalpies are used in conjunction with the energy-resolved mass spectrometry method of breakdown graphs to give a more complete quantitative aspect to the interpretation of the fragmentation processes. The dry eye condition affects millions of individuals world wide. The symptoms can be a result of simple irritation to the eye or a serious disease state. A dry eye model was developed using rabbits in order to study the compositional makeup of the tear components in hopes of identifying an underlying cause, or expressed effect of the dry eye condition. The major non-polar lipids of the tear were identified by mass spectrometry as mono and diacylglycerols, with a smaller extent of triacylglycerols. The major polar phosphorylated lipids were identified in the tear extract revealing that sphingomyelin based species were being expressed in the dry eye condition. The major proteins were determined to be the lower molecular weight lipid binding proteins where two specific species were found to increase in expression for the dry eye condition.

The possibilities of differentiating linkage positions and anomeric configurations of small oligosaccharides by negative ion mode MALDI using anion attachment followed by PSD are investigated. By careful initial adjustment of the focusing mirror ratios allowing acquisition of the peaks of interest within the same PSD segment, it is possible to obtain highly reproducible relative ion abundances. Discrimination of different linkage types is achieved by analysis of structurally-informative diagnostic peaks offered by PSD spectra of chloride adducts of oligosaccharides, whereas the relative peak intensities of selected diagnostic fragment pairs make differentiation of anomeric configuration possible. F- and Ac- cannot form anionic adducts with the oligosaccharides in significant yields. However, Br-, I- and NO3- anionic adducts consistently appear in higher abundances relative to [M - H]-, just like Cl-. Mildly acidic saccharides form both deprotonated molecules and anionic adducts, making it possible to simultaneously detect neutral and acidic oligosaccharides via anion attachment. PSD of [oligosaccharide + Cl]- yields structurally-informative fragment ions that retain the charge on the sugar molecule rather than solely forming Cl-, whereas PSD of Br-, I- and NO3- adducts of oligosaccharides yield the respective anions as the main product ions without offering structural information concerning the sugar. PSD of the chloride adduct of saccharides containing 1-2 linkages also yields chlorine-containing fragment ions. MALDI-TOF-MS and LDI-TOF-MS are shown to be useful for characterization of ultra-small titania nanoparticles. Peak maxima in MALDI-TOF mass spectra are found to correlate with nanoparticle size. The size distributions of TiO2 nanoparticles, obtained from MALDI- and LDI-TOF-MS are in good agreement with parallel TEM observations. PSD analysis of inorganic x nanomaterials is performed and valuable information about the structure of analytes has been obtained. A group of inorganic nitrate and perchlorate salts of forensic and health interest are investigated by LDI- and MALDI-TOF MS. In each case, a series of characteristic cluster ions are predominant in the negative-ion mode. The number and identity of metal atoms and anions in the recorded cluster ions can be positively identified by their m/z values, distinctive isotopic patterns and characteristic PSD fragmentation patterns.

Orientador: Ian Castro-Gamboa / Banca: Luiz Alberto Beraldo de Moraes / Banca: Jairo Kenupp Bastos / Banca: Daniel Rinaldo / Banca: Emerson Ferreira Queiroz / Resumo: A abordagem reducionista na busca por substâncias bioativas em produtos naturais nem sempre é capaz de compreender em sua totalidade a dinâmica envolvida nas respostas farmacológicas de matrizes complexas como extratos vegetais. Inserido neste paradigma, este trabalho visou ao desenvolvimento de métodos e abordagens racionais que permitissem o entendimento das relações moleculares em produtos naturais bioativos. Para tanto, espécies vegetais da família Chrysobalanaceae com potencial citotóxico, depositadas na extratoteca do NuBBE, foram os objetos de estudo na aplicação de metodologias para a determinação de sua composição metabólica e para a avaliação do potencial citotóxico. Os extratos hidroalcoólicos de Couepia grandiflora, Hirtella hebeclada, Licania hoehnei, Licania kunthiana, Licania huminis e Parinari excelsa foram analisados por CG-EM, CLAE-DAD-EM/EM e CLAE-autoEM/EM para a identificação in situ de sua composição metabólica, através de comparação dos dados espectrais a uma base de dados contendo valores de massas de alta resolução e espectros de UV de todos os metabólitos secundários já relatados para a família Chrysobalanaceae. O uso de ferramentas de análise multivariada, como a deconvolução empírica proposta pelo software AMDIS e a extração espectral desenvolvidas pelos algoritmos RAMSY e MCR-ALS, assistiram à desreplicação ao extrair os espectros dos compostos, mesmo em baixa resolução cromatográfica. A desreplicação das espécies de Chrysobalanaceae permitiu a detecção in situ de metabólitos primários (aminoácidos, ácidos e açúcares) e secundários (flavonoides-O-glicosilados e polímeros de proantocianidinas). Adicionalmente ao estudo de determinação metabólica das matrizes vegetais, realizaram-se ensaios de atividade citotóxica. Resultados preliminares da bioatividade ante diferentes linhagens de Saccharomyces cereviceae, obtidos... / Abstract: The reductionist approach for the search of bioactive compounds in natural products is not always being able to understand the complete dynamics involved in the pharmacological responses of complex matrices such as plant extracts. Based on this paradigm, this study aimed to develop rational methods approaches to runderstand the molecular relationships present in bioactive natural products. Chrysobalanaceae plant species with cytotoxic potential, deposited NuBBE's bank of extracts, were the objects of study in the application of methodologies for defining the metabolic composition and evaluate their cytotoxic potential. Couepia grandiflora, Hirtella hebeclada, Licania hoehnei, Licania kunthiana, Licania huminis and Parinari excelsa ethanol extracts were analyzed by GC-MS, HPLC-DAD-MS/MS and HPLC-autoMS/MS for in situ metabolic identification based on spectral comparison with a high resolution mass spectra and UV spectral database developed using previously reported data for Chrysobalanaceae. The use of multivariate analysis tools, the empirical deconvolution software AMDIS and the spectral extraction algorithms RAMSY and MCR-ALS assisted the dereplication by extracting spectral data for "pure" compounds even at low chromatographic resolution. The dereplication of Chrysobalanaceae species allowed the in situ detection of primary metabolites (amino acids, and sugars) and secondary (O-glycosylated-flavonoids and proanthocyanidins polymers). In addition to the metabolic determination of Chrysobalanaceae extracts, were performed cytotoxicity bioassays. Preliminary results of cytotoxicity against Saccharomyces cereviceae, carried out during the construction of the bank of extracts, indicated cytotoxic potential, especially for Hirtella hebeclada and Parinari excelsa. However, the cytotoxic assay against different tumor cell lines showed that the extracts were not active, which may be related to which may be... / Doutor

As an emerging field, lipidomics still encounters methodological challenges. Indeed, as it is an –omics field, analysis of data is time and labour intensive, if the necessary and appropriate bioinformatics tools are not existent. Here I present two programs I developed to address the challenges of peak identification and peak annotation and filtering for a targeted lipidomics approach, which is not supported by available software. The first program, Lipid Identification Tool (LIT), consists of a stand-alone offline search engine, which provides a robust in silico database generated by linear equations based on lipid structures, containing information lacking on presently available online databases. The second program, Lipid Identification for Targeted Lipidomics (LITL), allows the annotation of HPLC-ESI-MS/MS MRM acquired spectral data in text-based format, comparing them to a library of identities, via retention time and mass-over-charge of detected ions, and to easily export the results to a statistical analysis software. // La lipidomique est un jeune domaine encore proie à des défis quant à sa méthodologie. En effet, l’analyse de données en lipidomique demeurera longue et ardue, tant que des outils bioinformatiques appropriés ne sont pas créés. Je décris ici deux de mes programmes développés pour remédier aux défis d’identification de pics et d’annotations et de sélection de pics provenant d’une approche lipidomique dénommée ciblée, données présentement incompatibles avec d’autres programmes. Lipid Identification Tool (LIT) est un moteur de recherche hors-ligne possédant une robuste base de données in silico générée mathématiquement, et contenant des informations sur des espèces lipidiques non décrites dans les bases de données disponibles. Lipid Identification for Targeted Lipidomics (LITL) permet l’annotation de données provenant de méthodes HPLC-ESI-MS/MS MRM, en comparant leur temps de rétention ainsi que leur rapport masse-sur-charge à une bibliothèque d’identités lipidiques, et permet de les exporter facilement vers un logiciel d’analyse statistique.

A composição dos lipídeos de membrana está entre os fatores que podem influenciar a capacidade fecundante do espermatozoide. Sabidamente os lipídeos desempenham papéis indispensáveis na membrana dos espermatozoides, participando dos processos de interação entre gametas e influenciando o comportamento físico-químico dos espermatozoides durante os procedimentos de criopreservação. Estas moléculas apresentam uma ampla gama de propriedades químicas que tornam complexa a sua análise. Atualmente a espectrometria de massas (MS) por ionização e dessorção a laser assistida por matriz (MALDI) representa uma potente ferramenta para a análise de lipídeos. Assim, a proposta deste trabalho buscou verificar a ocorrência de diferença no perfil lipídico de espermatozoides bovinos obtidos por MALDI utilizado técnica de análise direta dos espermatozoides e análise após a extração lipídica pelo método de Bligh e Dyer. Além disso, este trabalho analisou o perfil lipídico de espermatozóides bovinos que receberam ou não suplementação de antioxidantes. Após coleta, lavagem e separação dos espermatozoide, estes foram submetidos à extração lipídica ou destinados íntegros à obtenção do perfil lipídico (método direto). O perfil lipídico do extrato lipídico ou de espermatozoides íntegros foi adquirido por MALDI em modo positivo e com matriz DHB (ácido dihidroxibenzoico). A análise dos dados foi realizado com o software Metaboanalist utilizando ferramentas de análise multivariada (análise de componentes principais - PCA) e de identificação de íons com intensidade diferencial entre grupos de comparação (teste t, volcano plot). Com essas análises foi possível identificar que ocorre diferença no perfil lipídico dos espermatozoides bovinos quando obtidos a partir de análise direta ou extração de lipídeos. Não foram observadas diferentes entre grupos submetidos ou não à suplementação com vitaminas. O método direto é capaz de proporcionar um perfil lipídico representativo e de fácil aquisição. / The membrane lipid composition is amongst the factors that influence the fertilization competence of the spermatozoa. It is also known that lipids play fundamental roles at spermatozoa membranes, participating in many processes, such as spermatozoaoocyte interaction and determining the physical-chemical behavior of spermatozoa membranes during cryopreservation. These molecules show a wide range of chemical properties, making complex its analysis. Currently, mass spectrometry (MS) through matrix-assisted laser desorption ionization (MALDI) represents a powerful tool for lipids analysis. Hence, this proposal aimed to evaluate differences in bovine spermatozoa lipid profile obtained through MALDI using direct sample analysis of spermatozoa or analysis of spermatozoa lipid extract obtained by Bligh-Dyer technique. Moreover, this proposal evaluated lipid profile of spermatozoa from bull that received or not antioxidant supplementation. After collection, washing and separation, the spermatozoa were submitted to lipid extraction or straightforward destined to lipid profile acquisition (direct method). The lipid profiles of spermatozoa lipid extracts or from whole spermatoloza were obtained by MALDI in positive mode with DHB matrix (dihydrozybenzoic acid). Data analysis was performed with Metaboanalyst software using multivariated analysis (Principal component analysis - PCA) and tools for identification of differential ions between comparison groups (t-test, volcano plot). Results show differences in spermatozoa lipid profile obtained by direct method or after lipid extraction. No differences were observed in bovine spermatozoa derived from bulls supplemented or not with vitamins. The direct method can generate a representative and easily obtained lipid profile.

Thesis advisor: Eranthie Weerapana / A chemical-proteomic platform to monitor cysteine sensitivity to transnitrosation Yani Zhou Dissertation advisor: Dr. Eranthie Weerapana Abstract S-nitrosation has emerged as a ubiquitous endogenous protein posttranslational modification that significantly impacts cellular protein function through a variety of mechanisms. Despite the advent of chemical and proteomic methods to study S-nitrosation, the subset of cellular cysteine residues that show uniquely high reactivity to endogenous transnitrosation donors is poorly characterized. To further these existing global studies, a cysteine-reactivity profiling strategy was applied herein to rank ~600 cysteine residues by sensitivity to S-nitrosoglutathione. These proteomic studies revealed several previously uncharacterized sites of S-nitrosation, including Cys58 in HADH2. Further characterization revealed that HADH2 catalytic activity is allosterically regulated by S-nitrosation, and this modification occurs in cells at (patho)physiological levels of nitrosative stress. Functional role of Cys58 and its regulation by S-nitrosation facilitated the identification of RB-21-CA as a potential covalent Cys58 inhibitor. Global analysis of GSNO, S-nitroso-Coenzyme A and Thioredoxin-C73-SNO transnitrosation identified 756 cysteines with different sensitivity to each of three SNO donors. Systematic evaluation on transnitrosation selectivity revealed that specific interaction of transnitrosation donor with its protein target is a key component governing the selective transnitrosation of a specific cysteine residue. Together, these studies illustrated the potential of cysteine-reactivity profiling strategy for evaluating the substrate specificity of transnitrosation donors and enable the identification of previously uncharacterized, functionally relevant sites of S-nitrosation. Another cysteine oxoform, S-glutathionylation, is the disulfide formation of a protein cysteine residue with glutathione. Although glucose starvation is known to induce redox-disturbance, global and individual protein S-glutathionylation in response to glucose metabolism or mitochondrial activity remains largely unknown. By using a clickable glutathione approach, which forms clickable glutathione by the use of a mutant of glutathione synthetase, we found that protein S-glutathionylation is readily induced in response to glucose starvation when mitochondrial reactive oxygen species are elevated in cells, and glucose is the major determinant for inducing reversible glutathionylation. Application of a proteomic mass spectrometry platform identified over 1,300 S-glutathionylated. Confirmation of S-glutathionylation for selected proteins by in gel analysis further validated the mass spectrometry results, and highlights the dynamic change of S-glutathionylation on an individual protein level. In order to expand on the understanding of the functional role of the cysteine residues in biological systems, we evaluated a panel of 1,3,5-triazine- and 4-aminopiperidine-based cysteine-reactive small-molecules on two proteins, apoptosis signal-regulating kinase 1 (ASK1) and peroxiredoxin 1 (Prdx1), between which a intermolecular disulfide forms and results in the activation of mitogen-activated protein kinase pathway. In-gel fluorescence revealed that RB-11-CA and SMC-1, both of which contain an n-octyl group as a diversity element, showed greatest selectivity and potency to ASK1 and Prdx1, respectively. Further mass spectrometry analysis identified cysteine 225 of ASK1 and cysteine 173 of Prdx1 are the sites of covalent probe modification. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.