Electrospray ionization mass spectrometry analysis of changes in phospholipids in RBL-2H3 mastocytoma cells during degranulation

Biological membranes contain an extraordinary diversity of lipids. Phospholipids function as major structural elements of cellular membranes, and analysis of changes in the highly heterogeneous mixtures of lipids found in eukaryotic cells is central to understanding the complex functions in which lipids participate. Phospholipase-catalyzed hydrolysis of phospholipids often follows cell surface receptor activation. Recently, we demonstrated that granule fusion is initiated by addition of exogenous, nonmammalian phospholipases to permeabilized mast cells. To pursue this finding, we use positive and negative mode Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to measure changes in the glycerophospholipid composition of total lipid extracts of intact and permeabilized RBL-2H3 (mucosal mast cell line) cells. The low energy of the electrospray ionization results in efficient production of molecular ions of phospholipids uncomplicated by further fragmentation, and changes were observed that eluded conventional detection methods. From these analyses we have spectrally resolved more than 130 glycerophospholipids and determined changes initiated by introduction of exogenous phospholipase C, phospholipase D, or phospholipase A2. These exogenous phospholipases have a preference for phosphatidylcholine with long polyunsaturated alkyl chains as substrates and, when added to permeabilized mast cells, produce multiple species of mono- and polyunsaturated diacylglycerols, phosphatidic acids, and lysophosphatidylcholines, respectively. The patterns of changes of these lipids provide an extraordinarily rich source of data for evaluating the effects of specific lipid species generated during cellular processes, such as exocytosis.

Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution. Addition of oligonucleotides resulted in formation of protein-oligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful too] in the study of protein-DNA interactions.

Method Development and Validation of Vitamin D2 and Vitamin D3 using Mass Spectrometry

Thesis (Master's)--University of Washington, 2016-06

A rapid HPLC-mass spectrometry cyclosporin method suitable for current monitoring practices

Objectives: Cyclosporin is an immunosuppressant drug with a narrow therapeutic window. Trough and 2-h post-dose blood samples are currently used for therapeutic drug monitoring in solid organ transplant recipients. The aim of the current study was to develop a rapid HPLC-tandem mass spectrometry (HPLC-MS) method for the measurement of cyclosporin in whole blood that was not only suitable for the clinical setting but also considered a reference method. Methods: Blood samples (50 mu L) were prepared by protein precipitation followed by C-18 solid-phase extraction while using d(12) cyclosporin as the internal standard. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface in positive ionization mode. Results: The assay was linear from 10 to 2000 mu g/L (r(2) > 0.996, n = 9). Inter-day,analytical recovery and imprecision using whole blood quality control samples at 10, 30, 400, 1500, and 2000 mu g/L were 94.9-103.5% and

Simultaneous determination of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone concentrations in rat serum by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry

An assay using high performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry (ESI-MS-MS) was developed for simultaneously determining concentrations of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone, in 50 mul samples of rat serum. Deuterated (d(3)) analogues of each compound were used as internal standards. Samples were treated with acetonitrile to precipitate plasma proteins: acetonitrile was removed from the supernatant by centrifugal evaporation before analysis. Limits of quantitation (ng/ml) and their between-day accuracy and precision (%deviation and %CV) were-morphine, 3.8 (4.3% and 7.6%); morphine-3-glucuronide, 5.0 (4.5% and 2.9%); oxycodone, 4.5 (0.4% and 9.3%); noroxycodone, 5.0 (8.5% and 4.6%). (C) 2004 Elsevier B.V. All rights reserved.

Dose-dependent modulation of the T cell proteome by ascorbic acid

To investigate the hypothesis that the micronutrient ascorbic acid can modulate the functional genome, T cells (CCRF-HSB2) were treated with ascorbic acid (up to 150 μM) for up to 24 h. Protein expression changes were assessed by two-dimensional electrophoresis. Forty-one protein spots which showed greater than two-fold expression changes were subject to identification by matrix-assisted laser desorption ionisation time of flight MS. The confirmed protein identifications were clustered into five groups; proteins were associated with signalling, carbohydrate metabolism, apoptosis, transcription and immune function. The increased expression of phosphatidylinositol transfer protein (promotes intracellular signalling) within 5 min of ascorbic acid treatment was confirmed by Western blotting. Together, these observations suggest that ascorbic acid modulates the T cell proteome in a time- and dose-dependent manner and identify molecular targets for study following antioxidant supplementation in vivo.

Improved sampling and detection of ignitable liquid residues and explosives by mass spectrometry and ion mobility spectrometry

Fire debris evidence is submitted to crime laboratories to determine if an ignitable liquid (IL) accelerant was used to commit arson. An ignitable liquid residue (ILR) may be difficult to analyze due to interferences, complex matrices, degradation, and low concentrations of analytes. Debris from an explosion and pre-detonated explosive compounds are not trivial to detect and identify due to sampling difficulties, complex matrices, and extremely low amounts (nanogram) of material present. The focus of this research is improving the sampling and detection of ILR and explosives through enhanced sensitivity, selectivity, and field portable instrumentation. Solid Phase MicroExtraction (SPME) enhanced the extraction of ILR by two orders of magnitude over conventional activated charcoal strip (ACS) extraction. Gas chromatography tandem mass spectrometry (GC/MS/MS) improved sensitivity of ILR by one order of magnitude and explosives by two orders of magnitude compared to gas chromatography mass spectrometry (GC/MS). Improvements in sensitivity were attributed to enhanced selectivity. An interface joining SPME to ion mobility spectrometry (IMS) has been constructed and evaluated to improve field detection of hidden explosives. The SPME-IMS interface improved the detection of volatile and semi-volatile explosive compounds and successfully adapted the IMS from a particle sampler into a vapor sampler. ^

Assessment of the occurrence and potential risks of pharmaceuticals and their metabolites in fish and water using liquid chromatography mass spectrometry

A comprehensive method for the analysis of 11 target pharmaceuticals representing multiple therapeutic classes was developed for biological tissues (fish) and water. Water samples were extracted using solid phase extraction (SPE), while fish tissue homogenates were extracted using accelerated solvent extraction (ASE) followed by mixed-mode cation exchange SPE cleanup and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 11 target pharmaceuticals analyzed, trimethoprim, caffeine, sulfamethoxazole, diphenhydramine, diltiazem, carbamazepine, erythromycin and fluoxetine were consistently detected in reclaimed water. On the other hand, caffeine, diphenhydramine and carbamazepine were consistently detected in fish and surface water samples. In order to understand the uptake and depuration of pharmaceuticals as well as bioconcentration factors (BCFs) under the worst-case conditions, mosquito fish were exposed to reclaimed water under static-renewal for 7 days, followed by a 14-day depuration phase in clean water. Characterization of the exposure media revealed the presence of 26 pharmaceuticals while 5 pharmaceuticals including caffeine, diphenhydramine, diltiazem, carbamazepine, and ibuprofen were present in the organisms as early as 5 h from the start of the exposure. Liquid chromatography ultra-high resolution Orbitrap mass spectrometry was explored as a tool to identify and quantify phase II pharmaceutical metabolites in reclaimed water. The resulting data confirmed the presence of acetyl-sulfamethoxazole and sulfamethoxazole glucuronide in reclaimed water. To my knowledge, this is the first known report of sulfamethoxazole glucuronide surviving intact through wastewater treatment plants and occurring in environmental water samples. Finally, five bioaccumulative pharmaceuticals including caffeine, carbamazepine, diltiazem, diphenhydramine and ibuprofen detected in reclaimed water were investigated regarding the acute and chronic risks to aquatic organisms. The results indicated a low potential risk of carbamazepine even under the worst case exposure scenario. Given the dilution factors that affect environmental releases, the risk of exposure to carbamazepine will be even more reduced.

Development of Advanced Capillary Electrophoresis Techniques with UV and Mass Spectrometry Detection for Forensic, Pharmaceutical and Environmental Applications

Capillary electrophoresis (CE) is a modern analytical technique, which is electrokinetic separation generated by high voltage and taken place inside the small capillaries. In this dissertation, several advanced capillary electrophoresis methods are presented using different approaches of CE and UV and mass spectrometry are utilized as the detection methods. Capillary electrochromatography (CEC), as one of the CE modes, is a recent developed technique which is a hybrid of capillary electrophoresis and high performance liquid chromatography (HPLC). Capillary electrochromatography exhibits advantages of both techniques. In Chapter 2, monolithic capillary column are fabricated using in situ photoinitiation polymerization method. The column was then applied for the separation of six antidepressant compounds. Meanwhile, a simple chiral separation method is developed and presented in Chapter 3. Beta cycodextrin was utilized to achieve the goal of chiral separation. Not only twelve cathinone analytes were separated, but also isomers of several analytes were enantiomerically separated. To better understand the molecular information on the analytes, the TOF-MS system was coupled with the CE. A sheath liquid and a partial filling technique (PFT) were employed to reduce the contamination of MS ionization source. Accurate molecular information was obtained. It is necessary to propose, develop, and optimize new techniques that are suitable for trace-level analysis of samples in forensic, pharmaceutical, and environmental applications. Capillary electrophoresis (CE) was selected for this task, as it requires lower amounts of samples, it simplifies sample preparation, and it has the flexibility to perform separations of neutral and charged molecules as well as enantiomers. Overall, the study demonstrates the versatility of capillary electrophoresis methods in forensic, pharmaceutical, and environmental applications.

Chemical Reaction Dynamics of the Liquid/Vapor Interface Studied by Mass Spectrometry

This thesis presents investigations of chemical reactions occurring at the liquid/vapor interface studied using novel sampling methodologies coupled with detection by mass spectrometry. Chapters 2 and 3 utilize the recently developed technique of field-induced droplet ionization mass spectrometry (FIDI-MS), in which the application of a strong electric field to a pendant microliter droplet results in the ejection of highly charged progeny droplets from the liquid surface. In Chapter 2, this method is employed to study the base-catalyzed dissociation of a surfactant molecule at the liquid/vapor interface upon uptake of ammonia from the gas phase. This process is observed to occur without significant modulation of the bulk solution pH, suggesting a transient increase in surface pH following the uptake of gaseous ammonia. Chapter 3 presents real-time studies of the oxidation of the model tropospheric organic compound glycolaldehyde by photodissociation of iron (III) oxalate complexes. The oxidation products of glycolaldehyde formed in this process are identified, and experiments in a deoxygenated environment identify the role of oxygen in the oxidation pathway and in the regeneration of iron (III) following photo-initiated reduction. Chapter 4 explores alternative methods for the study of heterogeneous reaction processes by mass spectrometric sampling from liquid surfaces. Bursting bubble ionization (BBI) and interfacial sampling with an acoustic transducer (ISAT) generate nanoliter droplets from a liquid surface that can be sampled via the atmospheric pressure interface of a mass spectrometer. Experiments on the oxidation of oleic acid by ozone using ISAT are also presented. Chapters 5 and 6 detail mechanistic studies and applications of free-radical-initiated peptide sequencing (FRIPS), a technique employing gas-phase free radical chemistry to the sequencing of peptides and proteins by mass spectrometry. Chapter 5 presents experimental and theoretical studies on the anomalous mechanism of dissociation observed in the presence of serine and threonine residues in peptides. Chapter 6 demonstrates the combination of FRIPS with ion mobility-mass spectrometry (IM-MS) for the separation of isomeric peptides.

Assessment of the Occurrence and Potential Risks of Pharmaceuticals and their Metabolites in Fish and Water Using Liquid Chromatography Mass Spectrometry

A comprehensive method for the analysis of 11 target pharmaceuticals representing multiple therapeutic classes was developed for biological tissues (fish) and water. Water samples were extracted using solid phase extraction (SPE), while fish tissue homogenates were extracted using accelerated solvent extraction (ASE) followed by mixed-mode cation exchange SPE cleanup and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 11 target pharmaceuticals analyzed, trimethoprim, caffeine, sulfamethoxazole, diphenhydramine, diltiazem, carbamazepine, erythromycin and fluoxetine were consistently detected in reclaimed water. On the other hand, caffeine, diphenhydramine and carbamazepine were consistently detected in fish and surface water samples. In order to understand the uptake and depuration of pharmaceuticals as well as bioconcentration factors (BCFs) under the worst-case conditions, mosquito fish were exposed to reclaimed water under static-renewal for 7 days, followed by a 14-day depuration phase in clean water. Characterization of the exposure media revealed the presence of 26 pharmaceuticals while 5 pharmaceuticals including caffeine, diphenhydramine, diltiazem, carbamazepine, and ibuprofen were present in the organisms as early as 5 h from the start of the exposure. Liquid chromatography ultra-high resolution Orbitrap mass spectrometry was explored as a tool to identify and quantify phase II pharmaceutical metabolites in reclaimed water. The resulting data confirmed the presence of acetyl-sulfamethoxazole and sulfamethoxazole glucuronide in reclaimed water. To my knowledge, this is the first known report of sulfamethoxazole glucuronide surviving intact through wastewater treatment plants and occurring in environmental water samples. Finally, five bioaccumulative pharmaceuticals including caffeine, carbamazepine, diltiazem, diphenhydramine and ibuprofen detected in reclaimed water were investigated regarding the acute and chronic risks to aquatic organisms. The results indicated a low potential risk of carbamazepine even under the worst case exposure scenario. Given the dilution factors that affect environmental releases, the risk of exposure to carbamazepine will be even more reduced.

IDENTIFICAÇÃO POLIFÁSICA DE GONGRONELLA SP. E DE RHIZOPUS SP. DEGRADADORES DO FUNGICIDA SISTÉMICO METALAXIL

A capacidade destes fungos degradarem compostos xenobióticos e recalcitrantes é uma característica biotecnologicamente importante tornando-os potencialmente úteis para processos de biorremediação. Gongronella sp. e Rhizopus sp. foram isolados do solo de vinhas da região do Alentejo, Portugal. Estes isolados mostraram elevada capacidade de degradarem o fungicida acilalanina metalaxil. No presente estudo, para a identificação polifásica de Gongronella sp. e Rhizopus sp., presuntivamente identificado como R. stolonifer, várias linhagens de referência da ordem Mucorales (Absidia, Circinella, Gongronella e Rhizopus) foram incluídas. A abordagem polifásica combinou a análise das macro- e micromorfologias, a análise da sequência inteira da região ITS ribossomal (i.e., ITS1/5.8S rDNA/ITS2) e ainda o uso da espectrometria de massas por Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF).

Polyimides from isomeric oxydiphthalic anhydrides

2,2',3,3'-Oxydiphthalic dianhydride (2,2',3,3'-ODPA) and 2,3,3',4'-ODPA were synthesized from 3-chlorophthalic anhydride with 2,3-xylenol and 3,4-xylenol, respectively. Their structures were determined via single-crystal X-ray diffraction. A series of polyimides derived from isomeric ODPAs with several diamines were prepared in dimethylacetamide (DMAc) with the conventional two-step method. Matrix-assisted laser desorption/ionization time-of-flight spectra showed that the polymerization of 2,2',3,3'-ODPA with 4,4'-oxydianiline (ODA) has a greater trend to form cyclic oligomers than that of 2,3,3',4'-ODPA. Both 2,2',3,3'-ODPA and 2,3,3',4'-ODPA based polyimides have good solubility in polar aprotic solvents such as DMAc, dimethylformamide, and N-methylpyrrolidone. The 5% weight-loss temperatures of all polyimides were obtained near 500 degreesC in air. Their glass-transition temperatures measured by dynamic mechanical thermal analysis or differential scanning calorimetry decreased according to the order of polyimides on the basis of 2,2',3,3'-ODPA, 2,3,3',4'-ODPA, and 3,3',4,4'-ODPA. The wide-angle X-ray diffraction of all polyimide films from isomeric ODPAs and ODA showed some certain extent of crystallization after stretching. Rheological properties revealed that polyimide (2,3,3',4'-ODPA/ODA) has a comparatively lower melt viscosity than its isomers, which indicated its better melt processability.

Design, synthesis and validation of an inhibitor of CGRP degradation

Introduction: In addition to their afferent role in detection and signalling noxious stimuli, neuropeptide-containing sensory nerves may initiate and maintain chronic inflammation in diseases such as periodontitis by an efferent process known as neurogenic inflammation. Neuropeptides are susceptible to cleavage by peptidases, and therefore, the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies in our laboratory showed that enzyme components of gingival crevicular fluid (GCF) from periodontitis sites selectively inactivated the neuropeptide calcitonin gene-related peptide (CGRP), known to have a role in inhibiting osteoclastic bone resorption. Objectives: The aim of this study was to design and synthesise a specific inhibitor to prevent the degradation of CGRP by components of GCF. Methods: A hydroxamate-based inhibitor with a biotinylated tag was designed to ensure selectivity for CGRP and ease of use for future purification strategies. The biotinylated peptide hydroxamate contained the P1-P4 amino acid sequence of the potential CGRP cleavage site and was synthesised by solid-phase methods using standard Fmoc chemistry. Inhibition of CGRP metabolism by GCF was determined by MALDI-mass spectrometry (MALDI-MS) using pooled GCF samples from periodontitis patients as a crude source of the CGRP-degrading enzyme. Results: MALDI-MS analysis of CGRP degradation showed almost complete inhibition in the presence of the biotinylated inhibitor. Our results showed that the rate-limiting step in the cleavage of CGRP is endopeptidase cleavage, followed by carboxypeptidase attack. Conclusion: This study demonstrates that the enzyme component of GCF responsible for the degradation of CGRP can be inhibited by a biotinylated hydroxamate modelled on a potential endopeptidase cleavage site. The biotin tag on the inhibitor will facilitate our future purification of the CGRP-cleavage enzyme using a streptavidin-agarose column.

Influence of glycosidic linkages and molecular weight on the fermentation of maltose-based oligosaccharides by human gut bacteria

A structure-function study was carried out to increase knowledge of how glycosidic linkages and molecular weights of carbohydrates contribute toward the selectivity of fermentation by gut bacteria. Oligosaccharides with maltose as the common carbohydrate source were used. Potentially prebiotic alternansucrase and dextransucrase maltose acceptor products were synthesized and separated into different molecular weights using a Bio-gel P2 column. These fractions were characterized by matrix-assisted laser desorption/ionization time-of-flight. Nonprebiotic maltooligosaccharides with degrees of polymerization (DP) from three to seven were commercially obtained for comparison. Growth selectivity of fecal bacteria on these oligosaccharides was studied using an anaerobic in vitro fermentation method. In general, carbohydrates of DP3 showed the highest selectivity towards bifidobacteria; however, oligosaccharides with a higher molecular weight (DP6-DP7) also resulted in a selective fermentation. Oligosaccharides with DPs above seven did not promote the growth of "beneficial" bacteria. The knowledge of how specific structures modify the gut microflora could help to find new prebiotic oligosaccharides.