Metabólitos secundários produzidos por actinobactérias são uma inesgotável fonte de compostos com potentes atividades biológicas e estruturas intrínsecas. O desenvolvimento em instrumentação analítica tem contribuído significantemente para acelerar o processo de identificação e caracterização desses metabólitos bioativos. Sem dúvida alguma, a espectrometria de massas (MS) e o seu acoplamento com técnicas de separação, especialmente a cromatografia líquida (UHPLC-MS), tem sido reconhecida como a técnica mais eficiente em análises de produtos naturais. Nesta dissertação foi explorado o potencial da espectrometria de massas como ferramenta analítica para a identificação e caracterização estrutural de fitotoxinas produzidas por actinobactérias isoladas da rizosfera de plantas da caatinga. Foram produzidos noventa extratos de actinobactérias, dos quais quinze apresentaram alguma atividade para o bioensaio da Lemna minor e seis apresentaram atividade para o bioensaio da Chlorella vulgaris. Os extratos brutos ativos das actinobactérias Caat 7-38, Caat 8-6 e Caat 5-29 foram selecionados para caracterização dos compostos ativos, os quais foram isolados empregando o fracionamento guiado por bioensaios. No extrato bruto Caat 7-38, a actinomicina D foi identificada como fitotoxina, ao passo que para o extrato bruto Caat 8-6, foi possível inferir a atividade fitotóxica à presença do griseorhodin A. Já para o extrato bruto Caat 5-29, o composto identificado com atividade fitotóxica apresenta uma estrutura inédita, provavelmente pertencente à classe das anguciclinonas. Foi realizado ainda um estudo para avaliar o efeito da adição de terras raras ao meio de cultivo da actinobacteria Caat 7-38. Para os meios de cultivos contendo neodímio e, principalmente, lantânio ocorreu uma superprodução da actinomicina D, indicando assim, o grande potencial da aplicação das terras raras nos estudos de micro-organismos. / Secondary metabolites produced by actinomycetes are an inexhaustible source of compounds with potent biological activities and intrinsic structures. The development analytical instrumentation has contributed significantly to accelerate the identification and characterization of these bioactive metabolites. Undoubtedly, mass spectrometry (MS) and its coupling with separation techniques, especially liquid chromatography (UHPLC-MS) has been recognized as the most \"efficient\" technique in natural product analysis. In this work was explored the potential of mass spectrometry as an analytical tool for identification and structural characterization of phytotoxins produced by actinomycetes isolated from the rhizosphere of plants from the Caatinga biome. Ninety actinomycetes extracts were produced, of which fifteen showed some activity for the bioassay with Lemna minor and six showed activity for the bioassay with Chlorella vulgaris. The crude active extract of actinomycetes Caat 7-38, Caat 8-6 and Caat 5-29 were selected to characterize the active compounds, which were isolated using bioassay-guided fractionation. In the crude extract Caat 7-38, actinomycin D was identified as phytotoxin, while for crude extract Caat 8-6, it was possible to infer phytotoxic activity to the presence of griseorhodin A. For the crude extract Caat 5-29, the compound identified with phytotoxic activity presents a new structure, probably belonging to the class of anguciclinones. A study to evaluate the effect of addition of rare earths to the culture medium of actinobacteria Caat 7-38 was also carried out. To the culture medium containing neodymium and especially lanthanum occurred overproduction of actinomycin D, thus indicating the great potential of application of rare earths in the studies of microorganisms.

Mass spectrometry is a versatile analytical tool for chemical and biomolecule identification, quantitation, and structural analysis. Tandem mass spectrometry further expands the applications of mass spectrometry, making it more than a mere detector. With tandem mass spectrometry, the mass spectrometer is capable of probing reaction mechanisms, monitoring reaction processes, and performing fast analysis on complex samples. In tandem mass spectrometry, after activation the precursor ions fragment into small fragment ions through one or more pathways, which are affected by the ion’s inherit property, the ion type, and the activation method. To obtain complementary information, one can alter the fragmentation pathway by changing the ion via ion charge manipulation and covalent modification to the ion. Gas-phase ion/ion reactions provide an easy approach to changing ion type and facile modification to the analyte ions. It has been extensively used for spectrum simplification and analyte structural studies. In this dissertation, ion/ion reaction facilitated mass spectrometry methods are studied, and explorations into the method development involving front-end mass spectrometer are discussed.<br>The first work demonstrates a special rearrangement reaction for gas-phase Schiff-base-modified peptides. Gas-phase Schiff-base modification of peptides has been applied to facilitate the primary structural characterization via tandem mass spectrometry. A major or minor fragment pathway related to the novel rearrangement reaction was observed upon in-trap collisional activation of the gas-phase Schiff-base-modified peptides. The rearrangement reaction involves the imine of the Schiff base and a nucleophile present in the polypeptide. The occurrence of the rearrangement reaction is affected by several factors, such as ion polarity, identity of the nucleophile in the peptide (e.g., side chains of lysine, histidine, and arginine), and the position of the nucleophile relative to the imine. The rearrangement reaction does not affect the amount of structural information that can be obtained by collisional activation of the Schiff-base-modified peptide, but when the rearrangement reaction is dominant, it can siphon away signal from the structurally diagnostic processes.<br>Efforts have also been put into the method development of peptide and protein aggregation detection via electrospray ionization mass spectrometry (ESI-MS). People have studied peptide and protein aggregation processes to understand the mechanism of amyloid-related diseases and to control the quality of the peptide and protein pharmaceuticals. ESI-MS is suitable for solution aggregation studies because of its compatibility with solution samples and the straightforward result of the analyte’s oligomeric state on the mass spectrum. However, peak overlap issue and nonspecific aggregation in the ESI process can obscure the result. Here, the application of proton transfer ion/ion reaction to the analyte has been found useful to reduce or eliminate the peak overlap issue. A statistical model based on Poisson statistics has been proposed to deal with the ESI-induced nonspecific aggregation in the droplet and to differentiate the solution-phase aggregation from the droplet-induced aggregation. Factors that affect the accuracy of the statistical model have been discussed with MATLAB simulations.<br>In the era of biological system studies, sample complexity is a challenge every analytical chemist has to face. The analysis of complex sample can be facilitated by the combination of separation techniques outside the mass spectrometer (such as differential mobility spectrometry (DMS)) and ion structure probing techniques inside the mass spectrometer (such as tandem mass spectrometry and gas-phase ion/ion reactions). Here the coupling method between DMS and ion/ion reaction is developed and tested with model peptide systems to demonstrate its possible application in complex sample characterization such as isomer identification.<br>

<div>Amongst the various classes of mass analyzers, the quadrupole ion trap (QIT) is by far the most versatile. Although it can achieve only modest resolution (unit) and mass accuracy (101-102 ppm), it has high sensitivity and selectivity, can operate at pressures exceeding 10-3 torr, is tolerant to various electrode imperfections, and has single analyzer tandem mass spectrometry (MS/MS) capabilities in the form of product ion scans. These characteristics make the QIT ideal for mass spectrometer miniaturization, as most of the fundamental performance metrics of the QIT do not depend on device size. As such, the current drive in miniature systems is to adopt miniature ion traps in various forms – 3D, linear, toroidal, rectilinear, cylindrical, arrays, etc.</div><div><br></div><div>Despite being one of the two common mass analyzers with inherent MS/MS capabilities (the other being the Fourier transform ion cyclotron resonance mass spectrometer), it is commonly accepted that the QIT cannot perform one-dimensional precursor ion scans and neutral loss scans - the other two main MS/MS scan modes - or two-dimensional MS/MS scans. The former two are usually conducted in triple quadrupole instruments in which a first and third quadrupole are used to mass select precursor and product ions while fragmentation occurs in an intermediate collision cell. The third scan can be accomplished by acquiring a product ion scan of every precursor ion, thus revealing the entire 2D MS/MS data domain (precursor ion m/z vs. product ion m/z). This, however, is not one scan but a set of scans. Because the ion trap is a tandem-in-time instrument rather than a tandem-in-space analyzer, precursor ion scans, neutral loss scans, and 2D MS/MS are, at best, difficult.</div><div><br></div><div>Yet miniature mass spectrometers utilizing quadrupole ion traps for mass analysis would perhaps benefit the most from precursor scans, neutral loss scans, and 2D MS/MS because they generally have acquisition rates (# scans/s) an order of magnitude lower than their benchtop counterparts. This is because they usually use a discontinuous atmospheric pressure interface (DAPI) to reduce the gas load on the backing pumps, resulting in a ~1 scan/s acquisition rate and making the commonly-used data-dependent acquisition method (i.e. obtaining a product ion scan for every abundant precursor ion) inefficient in terms of sample consumption, time, and instrument power. Precursor and neutral loss scans targeting specific molecular functionality of interest - as well as 2D MS/MS – are more efficient ways of moving through the MS/MS data domain and thus pair quite readily with miniature ion traps.</div><div><br></div><div>Herein we demonstrate that precursor ion scans, neutral loss scans, and 2D MS/MS are all possible in a linear quadrupole ion trap operated in the orthogonal double resonance mode on both benchtop and portable mass spectrometers. Through application of multiple resonance frequencies matching the secular frequencies of precursor and/or product ions of interest, we show that precursor ions can be fragmented mass-selectively and product ions ejected simultaneously, preserving their relationship, precursor ion -> product ion + neutral, in the time domain and hence allowing the correlation between precursor and product ions without prior isolation. By fixing or scanning the resonance frequencies corresponding to the targeted precursor and product ions, a precursor ion scan or neutral loss scan can be conducted in a single mass analyzer. We further show that 2D MS/MS - acquisition of all precursor ion m/z values and a product ion mass spectrum for every precursor ion, all in a single scan - is possible using similar methodology. These scan modes are particularly valuable for origin-of-life and forensic applications for which the value of miniature mass spectrometers is readily evident.</div>

Protein identification using tandem mass spectrometry (MS/MS) has proven to be an effective way to identify proteins in a biological sample. An observed spectrum is constructed from the data produced by the tandem mass spectrometer. A protein can be identified if the observed spectrum aligns with the theoretical spectrum. However, data generated by the tandem mass spectrometer are affected by errors thus making protein identification challenging in the field of proteomics. Some of these errors include wrong calibration of the instrument, instrument distortion and noise. In this thesis, we present a pre-processing method, which focuses on the removal of noisy data with the hope of aiding in better identification of proteins. We employ the method of binning to reduce the number of noise peaks in the data without sacrificing the alignment of the observed spectrum with the theoretical spectrum. In some cases, the alignment of the two spectra improved.

OBJECTIVE The purpose of this study was to examine the stability of a generic lansoprazole product in a 3 mg/mL sodium bicarbonate suspension under room temperature and refrigerated conditions. METHODS Lansoprazole suspensions (3 mg/mL) were prepared in triplicate using an 8.4% sodium bicarbonate vehicle for each storage condition (room temperature and refrigerated). During 1 month, samples from each replicate were periodically removed and analyzed for lansoprazole concentration by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Each sample was spiked with 10 mg/L omeprazole to serve as the internal standard. A positive electrospray LC-MS/MS method was validated over the calibration range of 5 to 25 mg/L using Food and Drug Administration Guidance. The identities of the analyte and internal standard in the samples were verified by monitoring the MS/MS transitions of m/z 370 to m/z 252 and m/z 346 to m/z 198 for lansoprazole and omeprazole, respectively. Additionally, the pH of the suspensions was monitored throughout the study. RESULTS The stability of lansoprazole in the oral sodium bicarbonate suspension under refrigeration is compromised prior to what has been previously reported in the literature. Samples kept at room temperature lost >10% of the lansoprazole after 48 hours compared with the refrigerated samples, which maintained integrity up to 7 days. No statistically significant difference was found between the pH of the room temperature and refrigerated suspension samples, indicating that this factor is not the cause for the differences in stability at these two conditions. CONCLUSIONS This study suggests that the extemporaneously compounded lansoprazole oral suspension prepared in 8.4% sodium bicarbonate should not be stored in plastic oral syringes longer than 48 hours at room temperature and no longer than 7 days when refrigerated. These data indicate an expiration time earlier than that previously reported for the refrigerated product (14 days).

Sphingolipids are found in essentially all animals, plants and fungi, and some prokaryotic organisms and viruses. Sphingolipids function as structural components of membranes, lipoproteins, and as cell signaling modulators and mediators. To complicate matters further, sphingolipids often vary in type in different regions of tissues, and even in single cells, the subcellular localization of sphingolipids and their metabolic enzymes, transport proteins and targets may influence their functions. It is important to study sphingolipids spatial distribution within living organisms to understand how sphingolipids are involved in complex biochemical processes. As part of this thesis, procedures were optimized for the use of matrix assisted laser desorption/ionization (MALDI) tissue mass spectrometry (TIMS) to visualize the location of several types of lipids including sulfatides (ST), gangliosides and phosphoglycerolipids in brains from a mouse model for Tay-Sachs/Sandhoff disease. MALDI-TIMS was next applied to human ovarian carcinoma tissue to detect sulfatide location and established that ST are associated specifically with the regions of the ovarian tissue that bear the carcinoma. Electrospray ionization tandem mass spectrometry (ESI-MS-MS) was also used to confirm that ST and galactosylceramide (GalCer) are elevated in ovarian cancer. Gene expression data using tumor cells collected using laser capture microdissection revealed greater expression of mRNAs for GalCer synthase, GalCer sulfotransferase (Gal3ST1) and other enzymes of ST biosynthesis in epithelial ovarian carcinoma cells. This is a unique combination of two complementary, profiling technologies--mass spectrometry (metabolomic approach) with analysis of gene expression to study complex cancer pathology. The next study focused on the subcellular location of sphingolipids. In comparison with wild type Hek293 cells, a Hek293 cell line stably overexpressing serine palmitoyltransferase (SPT1/2 cells) was found to have elevated amounts of all subspecies of ceramide (Cer), but produces disproportionately higher amounts of C18-Cer and GalCer. Since Cer is known to inhibit protein ER/Golgi trafficking, these studies found that the higher production of Cer caused impairment of ER/Golgi trafficking of Ceramide synthase 1 (CerS1), thus increased C18-Cer. In addition, since GalCer is only synthesized in the lumen of the ER, this impairement of ER/Golgi trafficking also gave GalCer synthase access to its substrate and increased GalCer biosynthesis. These studies illustrate the complexity of sphingolipid biology and the usefulness of multiple tools to understand sphingolipid complex biological processes.

Peptide a-amidation is known as a signature of bioactivity due to the fact that half of the bioactive peptides found in the nervous and endocrine systems are a-amidated and that most known a-amidated peptides are bioactive. a-Amidated peptides are produced by the oxidative cleavage of glycine-extended precursors. Peptidylglycine a-amidating monooxygenase (PAM) is the only known enzyme responsible for catalyzing this reaction and its sole physiological function is to convert glycine extended prohormones to their a-amidated forms. High levels of PAM are found in certain tissues with no corresponding level of amidated products suggesting the presence of undiscovered a-amidated peptide hormones. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for peptide identification due to its advantages of speed, sensitivity and applicability to complex peptide mixtures. Normally, spectra are interpreted using database search engines. However, database searching is inefficient and ineffective for the identification of endogenous peptide with post-translational modifications (PTM) due to its low identification rate and high demand for computing power. There is a specific mass difference of 58.0055 units between an a-amidated peptide and its corresponding C-terminal glycine-extended precursor. The two peptides will have similar chromatographic retention time and MS/MS fragmentation patterns resulting from the identical amino acids sequences except for relatively the small differences at the C-termini. Based on this, a new LC-MS/MS based strategy for screening for a-amidated peptides was developed. This strategy depends on PAM inhibition and the mass accuracy of mass spectrometry (< 3 ppm). The coexistence of a-amidated peptides and their C-terminal glycine-extended precursors was insured by growing cells in the presence of a PAM inhibitor. After LC-MS/MS, masses and retention times of parent ions were extracted from raw data files and scanned by a script for peptide pairs with similar retention times and a mass difference around 58.0055. Resulting pairs were further validated by comparing their fragmentation patterns in MS/MS spectra. Only peptide pairs that met all three criteria were considered for further interpretation. This reduced the number of MS/MS spectra requiring interpretation by >99% and, thus, enable the manual inspection of MS/MS for the candidate peptide pairs. A total of 13 a-amidated peptides were successfully identified from cultured mouse pituitary AtT-20 cells using this method and a few of these newly identified a-amidated peptides exhibited bioactivity. The adaptability of this strategy to screening for other PTMs is also discussed. Peptidylglycine a-hydroxylating monooxygenase (PHM) is one of PAM domains which can be expressed separately. It is a copper dependent enzyme that catalyzes the first step of the two-step peptide amidation reaction. Removal of the copper ions results in the loss of enzyme catalytic activity. A PHM based a-amidated peptide enrichment method was developed. This method includes two steps. First, cells grown in culture were treated with a PAM inhibitor to effect the cellular accumulation of glycine-extended peptides. In the second step, copper-depleted PHM (apo-PHM) was used to selectively bind glycine-extended peptides present in the cell extract. All other unbound peptides were removed during wash runs. apo-PHM was then reinstated with copper to convert bound glycine-extended peptides to hydroxylated peptides and release them. Hydroxylated product can be converted to a-amidated peptide under basic conditions. Experiments carried out using model glycine extended peptides showed a 40 – 120-fold enrichment using HPLC-fluorometric assay or MALDI-TOF quantification. This method proved successful when working with complex samples like cell extracts. The relative intensity of a known a-amidated peptide mouse joining peptide (mJP) from an AtT-20 extract was dramatically increased after enrichment experiments.

Matrix metalloproteinases (MMPs) network with other biological molecules to maintain the extracellular matrix (ECM) in normal physiology and perform different roles. Understanding and assigning specific role to each of 24 members of these endoproteinases is impeded because of lack of specific and efficient detection methods in biological samples. Moreover, MMP-based anti-cancer drug development has also been challenged because, currently, there is no robust methodology to distinguish the inactive pro-enzymes, active enzymes or those complexed with endogenous inhibitors in biological specimens. The objective of this project is to develop a chemical proteomics strategy based on Matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) to help identify and discriminate the various MMP forms. Firstly, a triazine dye-based ligand immobilized on chromatography beads was utilized to assess whether it binds to recombinant human MMPs (rhMMPs). The results highlighted that the ligand interacts with latent forms of MMPs in agreement with the literature. Secondly, the potential of the ligand was assessed using MALDI-MS/MS based methodology in in vitro cancer models. Cell line culture supernatants were used in amounts to emulate the availability of tumour biopsies in clinical settings. The MS/MS spectral peaks specific to MMPs (MMP-2 and MMP- 14), and two endogenous inhibitors TIMP-1 and TIMP-2 were found in affinity chromatography eluates of cell culture supernatants with higher Mascot scores for the latter. While western blot detected MMP-2 in cell extracts, MALDI-MS/MS did not detect MMPs because of amounts below the limit of detection (LOD) of the instrument. Although the ligand was found to be interacting with MMPs and detergent-free salt elution buffers improved MALDI analysis, recovery of MMPs from biological samples was sub-optimal. The dye ligand was observed to bind other enzymes and despite various strategies to reduce non-specific binding of proteins or enable selective elution did not improve MMP enrichment. Further work using methodology described in this study is required after scaling up the MMP amounts in biological specimen and to resolve the issue of non-specific binding of proteins to the ligand by understanding its structure.

Managed aquifer recharge (MAR) is a low-energy subsurface water treatment system with the potential of being an important component of sustainable water reuse schemes. Alongside common wastewater contaminants, MAR systems have been shown to attenuate a range of trace organic chemicals (TOrCs). Despite several factors being possibly important for TOrC attenuation, many have not been investigated in depth. This research effort investigated three factors affecting attenuation of the moderately degradable TOrCs: primary substrate, adaptation of the microbial community to presence of TOrCs, and groundwater temperature. The overall goal was to optimize TOrC attenuation using different MAR configurations considering how these factors affect TOrC attenuation. The primary substrate composition and concentration significantly impacted attenuation of the moderately degradable TOrCs. Lower primary substrate concentrations and more refractory carbon generally resulted in better TOrC transformation, a more diverse microbial community in the infiltration zone and more diverse capabilities for TOrC degradation. The enzyme group cytochrome P450 may be important for TOrC transformation since its genes were more abundant under carbon-starving primary substrate conditions. Adaptation of the microbial community by pre-exposure to TOrCs was not required in order to degrade them. However, adaptation to the primary substrate was necessary for TOrC biotransformation due to its effect on the microbial community. Attenuation of most TOrCs was unaffected by changes in temperature. Some moderately degradable TOrCs, however, were better attenuated at higher temperatures likely due to increased microbial activity. Others were better degraded at lower temperatures likely due to favorable sorption conditions. In the context of applying MAR systems to potential water reuse schemes within Saudi Arabia, a reconnaissance study of TOrC occurrence in treated wastewater effluents was undertaken. Most of the TOrCs targeted were detected at similar concentrations to US effluents at comparable plants. One of the plants studied, however, displayed a significantly different TOrC footprint from the other treatment plants due to the large number of international visitors in its sewershed. Findings from this occurrence study as well from other tasks provided inputs to a risk assessment framework to compare the effectiveness of MAR systems as part of a multiple-barrier water reuse scheme.

Low energy (tens of eV) polyatomic cations were used as probes for characterization of monolayers of spontaneously chemisorbed thiols on gold. Characteristics including chemical composition, surface order and orientation of the self-assembled monolayers (SAMs) can be derived by monitoring the products of projectile ion neutralization, surface-induced dissociation (SID), and ion-surface reactions.To study the influence of the terminal group chemical structures and orientations of the SAMs on ion-surface interactions, a series of semi-fluorinated alkane thiols with difluoromethylenes buried underneath hydrocarbon terminal groups were examined (CH3CF2CH2− and CH3CH2CF2−). Compared to terminally fluorinated SAMs, they showed more projectile ion neutralization and less internal to vibrational energy deposition into precursor ions. Projectile ion-hydrocarbon reactions decreased significantly when difluoromethylenes are one or two bonds away from the terminal group. Furthermore, ion-surface reaction results on surfaces with odd and even chain lengths suggested that they have similar terminal methyl orientations to their hydrocarbon counterparts.Mixed monolayers of CF3CF2(CH2)14SH (F-SAMs) and CH3(CH2)15SH (H-SAMs) with systematically changing electron transfer, energy deposition and ion-surface reaction were prepared using mixed thiols solution and micro-contact printing (μ-CP). The solution mixture system showed linear variations in electron transfer and energy deposition with different F-SAM surface concentrations, while non-linear changes occur for ion-surface reaction suggesting strong lateral interactions between the two components. These interactions are minimized in the μ-CP system containing domains of each thiol. Energy deposition on the patterned surfaces varies non-linearly with changing F-SAM concentration which differs from the homogenously mixed system.To explore SID with a 90 collision angle, eV SID of a series of protonated peptide ions were performed in an in-line sector Time-Of-Flight (TOF) mass spectrometer. The results were compared to keV collision-induced dissociation (CID) data collected with the same instrument. Fragmentation efficiency for SID was higher than CID for those peptides. In addition to the excellent control over laboratory collision energies with SID, different amount of energy deposition can be achieved when varying surface composition, e.g. using mixed F-SAM/H-SAM.Reactive ion scattering spectrometry (RISS) results provided more in-depth knowledge of low energy ion-surface interactions that will promote usage of RISS as a novel surface characterization technique.