Orientador: Rodrigo Ramos Catharino / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T08:28:48Z (GMT). No. of bitstreams: 1 Lima_EsteladeOliveira_D.pdf: 1997369 bytes, checksum: 4ea7a216a988228e6353f9295da72214 (MD5) Previous issue date: 2015 / Resumo: A Hanseníase é uma doença infecciosa e crônica causada pelo Mycobacterium leprae. Este patógeno infecta principalmente macrófagos e células de Schwann e apresenta alta distribuição pela pele e nervos periféricos. Clinicamente, a forma mais comum de identificação da doença é a presença de espessamento de nervos e lesões hipocrômicas com perda de sensibilidade, entretanto, 30% dos pacientes infectados podem não apresentar manifestação clínica típica. Atualmente, o teste considerado padrão ouro no diagnóstico da Hanseníase é a baciloscopia, diretamente dependente de biópsia de pele, um método invasivo e com baixa sensibilidade para as formas mais brandas da doença. O desenvolvimento de um método rápido, sensível e não invasivo seria de grande importância para o diagnóstico assertivo da hanseníase. Portanto, o objetivo deste trabalho foi identificar marcadores lipídicos em pacientes com Hanseníase diretamente a partir de "imprint" de pele, usando a espectrometria de massas como estratégia analítica. Para a coleta do "imprint" de pele, uma placa de sílica foi levemente pressionada contra a pele dos pacientes ou dos indivíduos saudáveis (grupo controle). Os lipídeos adsorvidos pela placa de sílica foram extraídos e submetidos à ionização por eletroctrospray e infusão direta em espectrômetro de massas de alta resolução (ESI-HRMS). Todas as amostras foram diferenciadas por análise estatística multivariada baseada na plataforma de lipidômica, o que ajudou a eleger marcadores de diferentes classes de lipídeos. As análises identificaram leucotrieno E4, glicosilceramida, fosfatidilserina, phthiocerol e ácido ?-smegma micólico como marcadores do grupo com hanseníase, diferentemente do grupo de indivíduos saudáveis, cujos marcadores identificados pertencem às classes de fosfolipídeos e gangliosídeos, próprios da constituição natural da pele. Os resultados encontrados indicam que o "imprint" de pele em placa de sílica, associado à ESI-HRMS, é promissor como método rápido e sensível para o diagnóstico da Hanseníase. Assim sendo, o método desenvolvido apresenta grande potencial para auxiliar na redução da cadeia de transmissão da Hanseníase, uma vez que quanto mais precoce for o diagnóstico, mais cedo se inicia o tratamento / Abstract: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which primarily infects macrophages and Schwann cells, presenting higher distribution on skin and peripheral nerves. Clinically, the most common form of identification is through the observation of anesthetic lesions; however, up to 30% of infected patients may not present this clinical manifestation. Currently, the gold standard diagnostic test for leprosy is based on skin lesion biopsy, which is invasive and presents low sensibility for suspect cases. Therefore, the development of a fast, sensible and noninvasive method that identifies infected patients would prove helpful for assertive diagnosis. The aim of this work was to identify lipid markers in leprosy patients directly from skin imprints, using a mass spectrometric analytical strategy. For skin imprint samples, a 1 cm2 silica plate was gently pressed against patients¿ or healthy volunteers¿ skin. Imprinted silica lipids were extracted and submitted to direct-infusion electrospray ionization high-resolution mass spectrometry (ESI-HRMS). All samples were differentiated using a lipidomics-based data workup employing multivariate data analysis, which helped electing markers from distinct lipid classes. Results indicated that phospholipds, sphingolipids and mycolic acids, which were absent in healthy control subjects, clearly presented different intensities when compared to patients¿ samples. Results indicate that silica plate skin imprinting associated with ESI-HRMS is a promising fast and sensible leprosy diagnostic method, even for patients without clinical skin manifestations. With an early leprosy diagnosis, an early and effective treatment can be feasible and thus the chain of leprosy transmission can be abbreviated / Doutorado / Fisiopatologia Médica / Doutora em Ciências

<div><div><div><div>Mass spectrometry is a widely used tool for efficient chemical characterization and identification. The development of electrospray ionization as a soft ionization method enables mass spectrometry for large biomolecule investigation. Protein as one of the most important classes of biomolecules, its structural changes including folding, unfolding, aggregation, degradation and post-translational modification all influence protein bioactivity. Protein characterization and identification are important for protein behavior mechanism understanding, which may further contribute to disease treatment development. Protein conformation changes are normally very fast, and the initial stages, which significantly influence the conformation changing pathway, normally occur in milliseconds or shorter time scale. Such a fast structural change is hard to be monitored using traditional bulk solution manipulations, and fast sample preparation methods are required. </div><div><br></div><div>In this thesis project, theta tips are applied as a microreactor and nESI-MS emitter to perform fast protein manipulation immediately before MS analysis. Theta tips can be operated in two modes. The first mode is for submillisecond time scale reactions. Proteins and reagents are loaded into different channels and directly sprayed out simultaneously. Proteins and reagents mix and react in the Taylor cone and subsequent droplets for submillisecond time scale. Through this method, pH induced protein folding is investigated and protein folding intermediates were captured. The second mode is for milliseconds or longer reactions. Differential voltages can be applied to each channel before ionization and spray. The electric field between the two channels induces in-tip electroosmosis, which lead to an in-tip mixing and reaction. In this mode, the reaction time is not limited by the droplet lifetime as in the first mode, but is controlled by electroosmosis time. By changing the electroosmosis square wave frequency and cycles, the mixing time can be elongated to milliseconds or longer, which is suitable for slower reaction study. </div><div>Joule heating is discovered during theta tip electroosmosis when samples are dissolved in buffer. The Joule heating effect is high enough to heat up the aqueous solution to at least 75 oC based on Raman thermometry measurement, while the actual peak temperature could be higher. The Joule heating effect in theta tip electroosmosis can be easily controlled by electroosmosis voltage, time, buffer concentration etc.. Proteins are thermally denatured by the Joule heating effect, and the denaturation extent correlates with Joule heating parameters. </div><div>With this results in hand, we are developing a protein melting temperature measurement method using theta tip Joule heating effect and mass spectrometry. This new melting temperature measurement method measures changes in protein mass and charge state distributions. Therefore, it could sensitively detect ligand loss and protein tertiary structural changes, which is an important compensation to current protein melting temperature measurement techniques like CD or DSC. Since the heating time is short and protein concentration for MS is low, protein aggregation and thermal fragmentation are highly avoided so a complete protein thermal unfolding process is monitored. Theta tip electroosmosis combining MS characterized protein thermal denaturation behavior from a new aspect.</div><div><br></div><div>Besides single protein folding and unfolding, protein identification and post-translational modification are important for proteomics study. The traditional bottom-up, top-down and middle-down methods are not able to both preserve intact protein mass and efficiently generate enough fragment peaks easily without performing gas phase dissociation. In this thesis, we also developed a new way to identify proteins combining limited trypsin digestion and mass spectrometry. Intact protein mass was preserved for protein size and PTM identification. Enough tryptic peptides were also generated for protein identification through database search.</div></div></div></div><div><br></div>

Presented in this thesis are two methods that are coupled to the instrumentation for the recovery and analysis of ultra-trace illicit drug residues. The electrostatic dust lifting process is coupled with nanomanipulation-nanospray ionization to retrieve drug particles off of hard surfaces for analysis. For the second method, drug residues from fingerprint impressions are extracted followed by analysis. The methodology of these hyphenated techniques toward forensic science applications is applied as to explore limits of detection, sensitivity, and selectivity of analytes as well as immediacy and efficiency of analysis. The application of nanomanipulation-coupled to nanospray ionization-mass spectrometry toward forensic science based applications is considered as future improvements to trace and ultra-trace analysis.

<div>This dissertation details three studies which utilize nontraditional applications of electrospray ionization mass spectrometry. The first study explores and discusses the limitations of identifying unknown drug metabolites using ion-molecule reactions performed inside a mass spectrometer and coupled with high performance liquid chromatography. Ultimately, it was concluded that some highly-efficient, MS² ion-molecule reactions coupled with some drug metabolites would be sufficiently sensitive for <i>in vivo</i> drug metabolism studies. However, this study also concluded that the rate of false-positives and false-negatives may be higher than previous publications suggest.</div><div><br></div><div>The next study analyzed sulfur-containing compounds under atypical negative mode electrospray ionization mass spectrometry conditions. After noting that low analyte flow rates during electrospray ionization experiments on ethanethiol resulted in significant oxygen incorporation, the aim of this study was to understand the chemistry behind the oxygen incorporation and search for ways to experimentally limit the degree of oxygen inclusion. The atypical conditions were ultimately shown to induce significant ozonolysis and other oxidation reactions. Ultimately, only the use of high flow rates or switching to a different ionization technique were successful in mitigating the oxidation product formation. A new reaction mechanism for the oxidation of ethanethiol with ozone was proposed. Quantum chemical calculations were used to support the mechanism.</div><div><br></div><div>Finally, electrospray ionization mass spectrometry was used to analyze mixtures of selenium and/or tellurium in amine-thiol solvent systems. Selenium and tellurium are essential components in many thin film solar cells and other photovoltaics and amine-thiol solvent systems have been identified as a key solution processing strategy for synthesizing selenium and tellurium thin films. However, the reaction between selenium/tellurium and the amine-thiol solvent system is poorly understood and requires detailed study before large-scale industrial synthesis can be achieved. In this study, the dissolution mechanisms for selenium and tellurium in two different amine-thiol solvent systems were explored and discussed. The role of the basicity of the amine, the relative concentrations of the thiol, and the presence of co-dissolved chalcogens were all studied and used to propose dissolution mechanisms. The results of the experiments were used to control the synthesis of lead-selenium-tellurium alloy nanoparticles and could inform further studies on controlling metal chalcogenide synthesis through the appropriate choice of amine-thiol solvents.<br></div>

<p> Mass spectrometry (MS) is one of the most commonly used analytical techniques in bioanalytical analysis, allowing scientists to characterize molecules with very diverse chemical features. The advance in ionization strategies significantly improves the potential in using MS for that purpose, especially electrospray ionization (ESI) can generate ions directly from solution in ambient conditions, showing high flexibility in coupling with other techniques. Moreover, a hallmark of the ESI of large polymeric molecules is also its tendency to generate a distribution of charge states based on their chemical characteristics, allowing us to exploit the multiple charging phenomenon in various applications. </p> <p>This dissertation introduces the relationships between ESI and multiple charging phenomena with different proposed ionization models, and how condensed-phase and gas-phase approaches affect the multiple charging phenomenon. Moreover, multiply charged ions permit gas-phase ion/ion reactions to occur without neutralizing the ions. Therefore, various ion/ion reactions can be utilized for distinct analytical purposes. Objectively, this dissertation focuses on the investigation of the multiple charging phenomenon from ESI-MS, and the applications from taking the multiply charged ions to perform gas-phase ion chemistry in order to a) manipulate the charges of the targeted ions; b) invert the polarity of the targeted ions; c) and characterization of the ions from the gas-phase ion/ion reactions.</p> <p> The first work demonstrates how multiple components (i.e., complicated mixtures) lead to a highly congested spectrum of ions with overlapped m/z values, resulting from the multiple charging phenomenon after the ESI process. Utilizing ionic reactions can de-congest the spectra via manipulating the charges of the ions to separate the overlapped signals. A universal spectral pattern in the ESI mass spectra is observed while analyzing multiply-charged homopolymers. Various parameters, such as the charges of the ions, widths of polymer distributions, monomer mass, and cationizing agent masses, are investigated to show how they can affect the appearance of the unique patterns, which condense the information of the overall distribution of the homopolymers. Combined with gas-phase charge reduction (i.e., proton transfer reaction), we can characterize the size distribution of polydisperse homopolymer samples.</p> <p>Second, a novel type charge inversion ion/ion reaction summarizing the conversion of multiply charged protein ions to their opposite polarity and still holds multiple charges is reported. The reaction occurs via a single ion/ion collision with highly charged reagent ions, which we usually obtain from biological relevant polymers. Hyaluronic acid (HAs) anions and polyethylenimine (PEI) cations are used as the charge inversion reagents to react with protein ions. Remarkably, inversion of high absolute charge (up to 41) from the reaction is demonstrated. All mechanisms for ion/ion charge inversion involve low-energy ions proceeding via the formation of a long-lived complex. Factors that underlie the charge inversion of protein ions to the opposite polarity with high charge states in reaction with those reagent ions are hypothesized to include: (i) the relatively high charge densities of the HA anions and PEI cations that facilitate the extraction/donation of multiple protons from/to the protein leading to multiply charged protein anions/cations, (ii) the relatively high sum of absolute charges of the reactants that leads to high initial energies in the ion/ion complex, and (iii) the relatively high charge of the ion/ion complex following the multiple proton transfers that tends to destabilize the complex.</p> <p>Third, shotgun MS strategies coupled with different gas-phase ion chemistry and tandem MS to analyze glycolipids are demonstrated. Glycolipids contain both carbohydrates and lipids structure components that it is incredibly challenging to analyze with MS. Isomeric cerebrosides (n-HexCer) and glycosphingosines (n-HexSph), which hold isomerisms in diastereomeric sugar head groups (glucose and galactose), anomeric glycosidic linkages (alpha- or beta-), and isomeric amide-bonded monounsaturated fatty acyl chain (double bond location) are successfully differentiated by dissociating gas-phase ion/ion reaction products, the charge-inverted complex cations. Both relative and absolute quantification of the isomers is also achieved, and analytical performances are evaluated in terms of accuracy, precision, and inter-day precision, allowing us to perform mixture analysis. Porcine brains were used to demonstrate the ability to profile and quantify those isomers from biological extracts. Moreover, a parallel workflow is also proposed for gangliosides, which have more complicated structures among their glycan moiety. Metal cation transfer, proton transfer, and charge inversion reactions are utilized to manipulate the ion types to provide better structural information. The proposed workflow allows us to clean up the mass spectra by neutralizing interfering isobaric ions, differentiate isomeric gangliosides, and perform relative quantitation when the standards are available. The workflow also is used to obtain gangliosides profiles from biological matrices. Overall, work in this dissertation takes advantage of the multiple charging phenomenon and couples with gas-phase ion/ion reactions to achieve various analyses among a wide range of biological-related samples.</p>

Analytical method validation is the process of establishing that an analytical technique is applicable for a proposed objective. Early in the method development of a new analytical technique an understanding of the instrumental components and procedures is elaborated through scientifically based optimization. The optimization experiments are used to define the operational parameters that yield the maximum performance by the analytical technique for the target analyte before commencing validation studies. This dissertation details method development through experimental investigations instrumental components of LEMS (substrate, laser parameters, and electrospray source conditions). Each instrumental component has a number of induvial parameters which are optimized to yield the maximum laser electrospray mass spectrometry (LEMS) signal intensity for a given analytical problem. LEMS uses a nonresonant, femtosecond (fs) laser to ablate analytes from a surface. Those ablated analytes are then captured by a perpendicular electrospray, ionized, and desolvated to produce ions which travel into the inlet of the mass spectrometer for analysis. Each element of the LEMS experimental setup works in a complementary fashion to generate a mass spectral signal which have specific optimization steps that can dramatically impact the data that can be acquired. The results of the optimization for each instrumental component will then be applied to preliminary method development experiments for the analysis of pharmaceutical compounds from complex formulations biomarker discovery for mice afflicted with a traumatic brain injury.The effect of the laser pulse duration on the ablation mechanism and amount of laser induced conformational changes of aqueous myoglobin was investigated using 55 fs, 56 picosecond (ps), and 10 nanosecond (ns) pulses and laser pulse energies from 0.05 to 1.6 mJ. It was found that the optical properties of the substrates (stainless-steel and quartz) and laser intensity regimes accessible by each pulse duration determined the amount of myoglobin ablated and subsequent mass spectral signal intensity. Laser ablation of myoglobin from both substrates using all laser pulse energies was observed for the 55 fs pulse while the 10 ns pulse required minimum pulse energies of 0.4 and 1.2 mJ for ablation of myoglobin to occur from stainless-steel and quartz, respectively. As the pulse duration increases, thermal processes increase which dictated the relative amount of protein unfolding, number of phosphate adducts, and degree of solvent adduction. Many of the common laser electrospray ionization (ESI) hybrid techniques employ ns pulse durations. However, the amount of ablated myoglobin originating from a ns pulse was observed to be dependent on the amount of energy that was absorbed by the substrate or sample. Experiments to increase the signal intensity while implementing ns laser electrospray mass spectrometry (ns-LEMS) were performed by exploiting the optical properties of nanomaterials as a potential matrix for desorption and detection of myoglobin. To estimate the contribution of the surface plasmon resonance (SPR) to the desorption of myoglobin under the different pulse duration regimes, the addition of an aqueous gold nanostar (GNS) matrix was implemented. GNSs have a SPR maximum of ~750 nm which overlaps strongly with the 780 nm laser wavelength. Gold nanospheres, which have a SPR of ~530 nm, have an absorption overlap 25 times less than that of the nanostars with the 785 nm laser light and therefore were chosen as a control gold nanoparticle matrix. It was observed that protein mixed with solution phase GNSs improved the laser ablation and consequent mass spectral signal intensity of the protein in comparison to both the nanosphere addition and ablation from quartz without nanomaterial addition for the 55 fs, 56 ps, and 10 ns pulses. This dissertation also extends to an investigation of the electrospray source and the roles that the nebulizing gas pressure, electrospray solution flow rate, and needle protrusion from the emitter sheath effects the electrospray analyte signal and stability. Interactions between the electrospray droplets and nebulizing gas were elucidated using an ablation chamber in which laser ablated analytes were carried via the nebulizing gas flow through the nebulizer sheath to interact with the electrospray Taylor cone, jet, and subsequent droplets. The signal intensity and relative standard deviation (RSD) of an infused Victoria blue solution was used to assess conventional ESI optimization experiments while a mixture of Gly-Gly-His, lactose, adenosine, and vitamin B12 was laser ablated within the ablation chamber for the optimization of the remote ablation device. It was found that a needle protrusion flush with the nebulizing sheath wall, 9 psi nebulizing gas pressure, and 9 µL/min ESI flow rate yielded the highest signal intensity for low and high mass analytes when utilizing the ablation chamber. However, the conventional ESI signal and stability was maximized using a needle protrusion of 0.6 mm from the sheath, 18 psi nebulizing gas pressure, and 9 µL/ min ESI flow rate. The last two chapters describe collaborative efforts with GlaxoSmithKline (GSK) and Temple University’s Lewis Katz School of Medicine with the application of LEMS to real world problems. The first of these chapters explores the preliminary method development results for sampling protocols of LEMS in a pathway to measuring the active ingredient in a formulation when differences in concentration are a percent or less for GSK. The results from the method development and optimization experiments in the previous chapters were applied to the GSK pharmaceutical manufacturing paradigm to test product quality in-line and in real-time instead of testing in a lab at the end of the manufacturing process. The LEMS sampling protocols involved ablation of either powder, compressed form, or solution containing powder using laser ablation. The ablated material was then entrained in an electrospray aerosol and transferred into a mass spectrometer for quantitative measurement of the molecules making up the powder, pill, or solution. Measurement time was on the order of seconds so that thousands of samples can be potentially measured in an hour. Future prospective experiments include additional optimization of the solution phase and compressed form sampling methods and, ultimately, the method validation of LEMS for quantifying active ingredients in pharmaceutical formulations. The last chapter seeks to develop new methods to map all biomarkers in traumatic brain injury (TBI) through mass spectrometry imaging (MSI), serum analysis, and protein derivatization assays. In this work, the Ramirez laboratory employs the controlled cortical impact model of experimental TBI in mice, harvests the brain (post injury) and prepares sections for analytical analysis. TBI is a complex injury involving multiple physiological and biochemical alterations to tissue. The potentially thousands of relevant biomarkers spread over a volume of thousands of mm3 makes the spatially resolved chemical analysis of brain a big data problem to which principal component analysis is applied. / Chemistry

In this thesis the fabrication and analytical evaluation of two new electrospray emitters utilized for mass spectrometry analysis is presented. The emitters are based on a new concept, where the spray orifice can be varied in size. The thesis is based on two papers. All present-day nanoelectrospray emitters have fixed dimensions. The range of the applicable flow rate for such an emitter is therefore rather limited and exchange of emitters may be necessary from one experiment to another. Optimization of the signal of the analyte ions is also limited to adjustments of the applied voltage or the distance between the emitter and the mass spectrometer inlet. Furthermore, clogging can occur in emitters with fixed dimensions of narrow orifice sizes. In this thesis, electrospray emitters with a variable size of the spray orifice are proposed. An open gap between two thin substrates is filled with sample solution via a liquid bridge from a capillary. Electrospray is generated at the end point of the gap, which can be varied in width. In Paper I, electrospray emitters fabricated in polyethylene terephthalate have been evaluated. Triangular tips are manually cut from the polymer film. The tips are mounted to form a gap between the edges of the tips. The gap wall surfaces are subjected to a hydrophilic surface treatment to increase the wetting of the gap walls. In Paper II, silicon electrospray chips with high precision are fabricated and evaluated. A thin beam, elevated from the bulk silicon chip is fabricated by means of deep reactive ion etching. The top surfaces of the beams of two chips act as a sample conduit when mounted in the electrospray setup. An anisotropic etching step with KOH of the intersecting &lt;100&gt; crystal planes results in a very sharp spray point. The emitters were given a hydrophobic surface treatment except for the hydrophilic gap walls. For both emitter designs, the gap width has been adjusted during the experiments without any interruption of the electrospray. For a continuously applied peptide mixture, a shift towards higher charge states and increased signal to noise ratios could be observed when decreasing the gap width. The limit of detection has been investigated and the silicon chips have been interfaced with capillary electrophoresis. / QC 20101108

No description available.

Electrospray ionization (ESI) has become one of the most commonly used ionization techniques for mass spectrometry (MS) and ion mobility spectrometry (IMS), and efforts continue to improve its performance. ESI-MS is most recognized for its wide application to biomacromolecules where high sensitivity is of paramount importance. However, the major limitation in sensitivity with ESI-MS is due to its low ion transmission efficiency from the ESI source into the sampling orifice and through any stages utilized for transfer of ions from atmosphere to vacuum in the MS. A series of atmospheric pressure ion focusing interfaces were designed and implemented to enhance the performance of ESI-MS. The technical objective of this work was to improve sensitivity and detection limits of ESI-MS using a combination of concentric high velocity converging gas flow (aerodynamic focusing) and regulated external electric field (electrostatic focusing) to assist in focusing and transporting ions from the ESI sprayer tip into the sampling nozzle of the MS. The separation time in IMS, based on differing gas phase ion mobilities, ranges from several hundred microseconds to milliseconds. This allows faster analysis than most other conventional separation techniques, such as gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). However, the major limitation in ESI-IMS is its low resolution. It is believed that one of the most important contributions to low resolution in ESI-IMS is unwanted ion penetration through the ion gate. In order to solve this ion penetration problem, two mechanical ion gates were designed and optimized to assist in gating sprayed ions from the ESI source into the drift region of the IMS with improved sensitivity and resolution at atmospheric pressure. Applying a voltage to the ion gate and using a high flow drift gas helped to further improve the performance of ESI-IMS. Reduced pressure IMS should help to eliminate clustering and multiple peaks and, hence, improve experimental resolution when using ESI. Therefore, I report the design, construction and evaluation of new IMS systems for reduced pressures. However, the performance of the reduced pressure IMS was not as good as when using atmospheric pressure IMS.

Desorption electrospray ionization (DESI) is an ambient ionization technique for mass spectrometry. This solvent based desorption ion source has wide applicability in surface analysis with minimal sample preparation. Interest in improving detection limits, broadening applications, and increasing the spatial resolution for chemical imaging has led to studies of the DESI mechanism. An inverted microscope has been used to image interactions between the DESI spray and test analytes on a glass surface. Microscopic images recorded with millisecond time resolution have provided important insights into the processes governing analyte transport and desorption. These insights are the basis of a rivulet-based model for desorption that differs significantly from the widely-accepted momentum transfer model.