The purpose of the studies included in this thesis is to develop an effective an efficient method to study the proteome using separation and detection of peptides, when only a limited amount of sample, 10 micrograms of total protein or less, is available. The analysis will be applied to multiple myeloma cancer cells using ultra high-performance liquid chromatography-mass spectrometry for expression proteomics to illustrate utility. To detect low abundance peptides in a complex proteome, we use different strategies, including basic pH reversed-phase liquid chromatography (bRPLC), mass-to-charge fractionation in the mass spectrometer, and various liquid chromatography gradients to increase peptide separation to improve opportunities for detection and quantification. The different methods are optimized and compared by the number of peptides detected. Step-wise elution of bRP spin columns proved to yield more than 36,000 peptides using only 10 μg of protein. Mass-to-charge (m/z) fractionation was tested in mass analyzer Q-Exactive Plus (Thermo Scientific). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of an unfractionated sample was analyzed 4 times at different mass ranges, each mass range width of 150 m/z, resulting from 4 spectra combined, 31,732 peptides representing 3,967 proteins. Showingcomparable results to those form high pH reversed phase fractionation spin columns 5 fractions. Establishing a benchmark where the LC-MS/MS analysis of 600 μg of 10plex TMT-labeled peptides fractionated with bRPLC into 24 fractions yielded over 74,000 peptides from 7,700 proteins, we compared those results with analysis of 10 μg of total TMT-labeled peptides fractionated by bRP spin columns into 5 fractions, which produced 14,019 peptides from 3,538 proteins. These experiments were used to relatively quantify protein expression in naïve and drug resistant multiple myeloma cells lines as an example application in cancer research.

Phäochromozytome und Paragangliome (PPGL) sind seltene, katecholaminproduzierendeTumore des chromaffinen Gewebes. Die Erkrankung ist durch die Überproduktion von Katecholaminen gekennzeichnet und kann lebensbedrohliche Folgen haben. Die dieser Arbeit zugrunde liegende Studie untersuchte die interindividuellen Unterschiede im Metabolitenprofil bei Patient*innen mit PPGL im Vergleich zu Kontrollen mittels Flüssigchromatographie-Massenspektrometrie und einem Targeted Metabolomic Ansatz. Targeted Metabolomics beschreibt die Messung und Quantifizierung von im Voraus definierten Metaboliten in einer Probe. Von den 188 gemessenen Metaboliten zeigten vier Metabolite eine signifikanten Veränderung zwischen den Gruppen (Histidin, Threonin, LysoPC a C28:0 und Summe der Hexosen). Für alle vier Metabolite wurde ein Zusammenhang mit Katecholaminen im Urin beziehungsweise Metanephrinen im Plasma nachgewiesen. Subgruppenanalysen zeigten weitere Hinweise auf geschlechts- und phänotypspezifische Unterschiede im Metabolitenprofil zwischen Patient*innen mit PPGL und Kontrollen. / Pheochromocytomas and paragangliomas (PPGL) are rare, catecholamine-producing tumors arising from chromaffin cells. The disease is characterized by the overproduction of catecholamines and can have life-threatening consequences. The study on which this work is based investigated the interindividual differences in metabolite profiles in patients with PPGL compared to controls using liquid chromatography-mass spectrometry and a targeted metabolomics approach. Targeted metabolomics describes the measurement and quantification of predefined metabolites. Of the 188 metabolites measured, four metabolites showed a significant change between groups (histidine, threonine, LysoPC a C28:0 and sum of hexoses). A significant correlation with urinary catecholamines and/ or plasma metanephrines was identified for this metabolites. Subgroup analyses showed further evidence of sex- and phenotype-specific differences in metabolite profiles between patients with PPGL and controls.

<p>Metabolites, small endogenous molecules existing in every living cell, tissue or organism, play a vital role for maintaining life. The collective group of all metabolites, the metabolome, is a consequence of the biochemistry and biochemical pathways that a cell or tissue uses to promote survival. Analysis of the metabolome can be done to reveal changes of specific metabolites which can be a manifestation, a reason or a consequence of for example a disease. The physical chemical diversity amongst these components is tremendous and it poses a large analytical challenge to measure and quantify all of them. Targeting sub groups of the meta­bolome such as specific functional classes has shown potential for increasing metabolite coverage. Group selective labeling with biotin-tags followed by high affinity avidin capture is a well established purification strategy for protein purification.</p><p>The purpose with this project is to explore if it is possible to transfer the avidin biotin approach to metabolomics and use this method for small mole­cules purification. Specifically, this investigation aims to see if it is achievable to make a bio­tinylation of specific functional groups, to increase the sensitivity through reduction of sample complexity in liquid chromatography mass spectrometry metabolomics analyses after high affinity avidin capture. By purifying the analyte of interest and thereby reducing the sample complexity there will be a reduction in ion suppression. The aim is to increase the analytical sensitivity through a reduction in ion suppression during liquid chromatography mass spectrometry analysis.</p><p>Delimitations have been done to only investigate the possibility to obtain a biotinylation of primary amines and amides. As model compounds phenylalanine, spermi­dine, histamine and nicotinamide have been selected.</p><p>The result from this study indicates that it is possible to increase metabolite coverage through biotin labeling followed by high affinity avidin capture. It is a gain in analytical sensitivity of selected model compounds when comparing biotinylation strategy with a control non­biotinylation approach in a complex sample. A broader study of additional model compounds and a method development of this strategy are necessary to optimize a potential future method.</p>

The field of proteomics encompasses the study of identities, interactions, and dynamics of all proteins expressed by a living system. Research in this dissertation blends biochemical and quantitative proteomics techniques to increase the latitude of biological applications for the bottom-up mass spectrometry proteomics approach. Together, isolation of selected protein “targets,” such as multiprotein complexes, and quantitative characterization yields information essential for more detailed understanding of microbial cell function. Often, a challenging aspect of characterizing a variety of biochemically enriched samples is limited protein yield. This dissertation describes an enzymatic proteolysis protocol employing an organic/aqueous solvent that alleviates excessive handling steps to reduce losses during sample preparation for small quantities of protein samples. Presence of artifactual, non-specific proteins in enriched protein complex isolates complicates biological interpretation of specific protein interactions. Heterologous expression of affinity-tagged bait proteins may also cause unintended collateral effects. A series of local and global protein isotope ratio measurements were performed to differentiate authentic interactions from artifactual interactions among affinity-isolated complexes and assess collateral effects, respectively. Protein localization provides clues regarding protein function. To infer protein localization, quantitative proteomics techniques were used to estimate protein enrichment of cold osmotic shock periplasmic isolates. Protein isotope ratios indicating enrichment, combined with identification of amino-terminal signal peptide cleavages, increase confidence of periplasmic localization. Collectively, this dissertation provides a framework for tailoring biochemical and quantitative techniques for targeted characterization of microbial protein isolates.

Antimicrobials administered to livestock can be excreted up to 80% in the feces and urine. Liquid swine manure from confined animal feeding operations is generally retained in lagoon storage until it is applied as a nutrient source to cropland. Thus, the applied manure becomes a possible source of antimicrobials to aquatic ecosystems. Veterinary antimicrobials have been detected in surface and ground waters in Canada, the United States and Europe, however, their environmental fate is not well known. Lincomycin and spectinomycin are two antimicrobials administered as a mixture to swine in the prairie region of Canada for the prevention of post-weaning diarrhea. In order to assess the potential for contamination of prairie wetlands, concentrations of both antimicrobials were monitored in the liquid manure from the nursery area of a commercial-scale barn during a 5-week study, and their persistence during simulated manure storage investigated. The potential for transport of lincomycin and spectinomycin to surface waters via surface runoff and to leach to groundwater was also assessed. This was achieved by monitoring manure-amended soil, simulated rainfall runoff, snow melt runoff and groundwater over a two-year period at two study sites in Saskatchewan, Canada following fall application of liquid swine manure from two commercial barns to crop and pasture land. Liquid chromatography coupled with tandem mass spectrometry was used to quantitate these antimicrobials in all matrix extracts. <p>In the nursery area of a commercial-scale barn, concentrations of lincomycin and spectinomycin in the cumulating liquid manure at the end of the study were equivalent to 32 and 3.0%, respectively, of doses administered in the feed. In a laboratory study, using fortified liquid manure, concentrations of both antimicrobials showed a rapid initial decrease during simulated lagoon storage, followed by a slower dissipation over a period of 5 months. The average time required for 50% dissipation of lincomycin was greater than one year (365 d) and was approximately 90 d for spectinomycin. <p>Lincomycin concentrations in soil (46.3 to 117 µg kg-1) collected immediately after fall manure application, decreased to non-detectable levels by mid-summer the following year. Lincomycin was present in simulated rainfall runoff (0.1 to 2.7 µg L-1) immediately after manure application with similar concentrations present in snow meltrunoff the following spring. Concentrations in groundwater were generally <0.005 µg L-1. Spectinomycin was not detected in the manure applied at the study sites nor in soil, runoff water or groundwater samples. This study confirms that some antimicrobials, including lincomycin, may be present in lagoon manure. Thus, the management practice of utilizing livestock manure from confined animal feeding operations as a plant nutrient source on cropland may result in antimicrobial transport to surface and ground waters.

Hereditary Hemochromatosis (HH) is an inherited recessive autosomal disorder characterized by accumulation of excess iron. When iron binding proteins become saturated, concentrations of free, or non-transferrin-bound iron (NTBI) rise, a condition thought to be responsible for the adverse effects associated with HH. To investigate that disturbing iron homeostasis plays a role in free radical injury in HH, protein carbonyls were found to be 1-7 times higher in patients with HH than in controls, with the greatest increases being observed in untreated HH patients with high ferritin and >90% transferrin saturation with iron. An Unpaired t test revealed a P value of 0.0278 (P< 0.05), which is considered to be statistically significant. Our data showed a significant positive correlation (linear relationship) between the level of carbonyl content and ferritin concentration in plasma samples from patients with HH. In vitro oxidation of transferrin and ferritin standards with hydrogen peroxide and excess iron, followed by immobilized trypsin digestion (Poroszyme), high-resolution LC-MS/MS analysis (Q-TOF Ultima, Waters) and MS/MS data processing (PEAKS, Bioinformatics Solution), identified several tryptic peptides containing oxidized Met,Trp and His residues. Mapping of the oxidized ferritin residues showed them to be located on the inner face of each sub-unit, the face directed toward the ferritin core where iron is normally stored. Using the same methodology, oxidized residues were subsequently detected in ferritin and transferrin isolated from plasma samples of patients severely affected with HH. Comparing of MS/MS spectra of in vitro oxidized samples that have most fragment ion peaks in common with oxidized peptide MS/MS spectra from samples of patients with HH revealed a significant correlation between the two. These data show that elevated NTBI may be involved in oxidative modification of the iron binding proteins, ferritin and transferrin, and that such modifications may play a significant role in the pathophysiology of HH.

When exposed to them, isocyanates can induce serious injuries in the respiratory tract and irritation on the skin and in the eyes. They are therefore interesting from the point of view of occupational health. The purpose of this thesis was to collect isocyanates in air with impinger-filter samplers and solvent free samplers. Furthermore the isocyanates were to be analyzed with liquid chromatography-mass spectrometry. The solvent free sampler consists of a polypropylene tube and filter holder fitted with glass fiber filters impregnated with derivatization reagent, coupled with a pump. The impinger-filter sampler was made out of an impinger flask, containing a derivatization solution, coupled in series with a filter holder and a pump. Di-n-butylamine was used as derivatization reagent in both samplers to stabilize the reactive isocyanates and to enable mass spectrometric detection. The solvent free sampler is highly advantageous because of its user friendliness during field measurements, as opposed to the impinger-filter method. An exposure chamber, equipped with two interior fans to ensure good circulation, was used to generate an atmosphere containing isocyanates. Analyzing the isocyanate-DBA derivates with LC-MS/MS worked very well and the method made it possible to detect isocyanate levels below 1 ng/mL. During quantification of isocyanates a standard curve with concentrations from 1 to 1000 ng/mL was used. Detection of isocyanate levels as low as one fifth of the limit value of some isocyanates was found to be possible, which would indicate that the methods are sensitive. Even though the solvent free sampler worked, the impinger-filter sampler was found to be more effective in collecting isocyanates. The coefficient of variation calculated from concentrations of isocyanates from the solvent free sampler varied between 0-35 %. The reason for this might be due to the fact that an optimized extraction method had not been tried out within the time limit of this project. Questions, identified during this thesis work, need to be answered before being able to obtain reliable results from field measurements with the solvent free sampler.

Antimicrobials administered to livestock can be excreted up to 80% in the feces and urine. Liquid swine manure from confined animal feeding operations is generally retained in lagoon storage until it is applied as a nutrient source to cropland. Thus, the applied manure becomes a possible source of antimicrobials to aquatic ecosystems. Veterinary antimicrobials have been detected in surface and ground waters in Canada, the United States and Europe, however, their environmental fate is not well known. Lincomycin and spectinomycin are two antimicrobials administered as a mixture to swine in the prairie region of Canada for the prevention of post-weaning diarrhea. In order to assess the potential for contamination of prairie wetlands, concentrations of both antimicrobials were monitored in the liquid manure from the nursery area of a commercial-scale barn during a 5-week study, and their persistence during simulated manure storage investigated. The potential for transport of lincomycin and spectinomycin to surface waters via surface runoff and to leach to groundwater was also assessed. This was achieved by monitoring manure-amended soil, simulated rainfall runoff, snow melt runoff and groundwater over a two-year period at two study sites in Saskatchewan, Canada following fall application of liquid swine manure from two commercial barns to crop and pasture land. Liquid chromatography coupled with tandem mass spectrometry was used to quantitate these antimicrobials in all matrix extracts. <p>In the nursery area of a commercial-scale barn, concentrations of lincomycin and spectinomycin in the cumulating liquid manure at the end of the study were equivalent to 32 and 3.0%, respectively, of doses administered in the feed. In a laboratory study, using fortified liquid manure, concentrations of both antimicrobials showed a rapid initial decrease during simulated lagoon storage, followed by a slower dissipation over a period of 5 months. The average time required for 50% dissipation of lincomycin was greater than one year (365 d) and was approximately 90 d for spectinomycin. <p>Lincomycin concentrations in soil (46.3 to 117 µg kg-1) collected immediately after fall manure application, decreased to non-detectable levels by mid-summer the following year. Lincomycin was present in simulated rainfall runoff (0.1 to 2.7 µg L-1) immediately after manure application with similar concentrations present in snow meltrunoff the following spring. Concentrations in groundwater were generally <0.005 µg L-1. Spectinomycin was not detected in the manure applied at the study sites nor in soil, runoff water or groundwater samples. This study confirms that some antimicrobials, including lincomycin, may be present in lagoon manure. Thus, the management practice of utilizing livestock manure from confined animal feeding operations as a plant nutrient source on cropland may result in antimicrobial transport to surface and ground waters.

Hereditary Hemochromatosis (HH) is an inherited recessive autosomal disorder characterized by accumulation of excess iron. When iron binding proteins become saturated, concentrations of free, or non-transferrin-bound iron (NTBI) rise, a condition thought to be responsible for the adverse effects associated with HH. To investigate that disturbing iron homeostasis plays a role in free radical injury in HH, protein carbonyls were found to be 1-7 times higher in patients with HH than in controls, with the greatest increases being observed in untreated HH patients with high ferritin and >90% transferrin saturation with iron. An Unpaired t test revealed a P value of 0.0278 (P< 0.05), which is considered to be statistically significant. Our data showed a significant positive correlation (linear relationship) between the level of carbonyl content and ferritin concentration in plasma samples from patients with HH. In vitro oxidation of transferrin and ferritin standards with hydrogen peroxide and excess iron, followed by immobilized trypsin digestion (Poroszyme), high-resolution LC-MS/MS analysis (Q-TOF Ultima, Waters) and MS/MS data processing (PEAKS, Bioinformatics Solution), identified several tryptic peptides containing oxidized Met,Trp and His residues. Mapping of the oxidized ferritin residues showed them to be located on the inner face of each sub-unit, the face directed toward the ferritin core where iron is normally stored. Using the same methodology, oxidized residues were subsequently detected in ferritin and transferrin isolated from plasma samples of patients severely affected with HH. Comparing of MS/MS spectra of in vitro oxidized samples that have most fragment ion peaks in common with oxidized peptide MS/MS spectra from samples of patients with HH revealed a significant correlation between the two. These data show that elevated NTBI may be involved in oxidative modification of the iron binding proteins, ferritin and transferrin, and that such modifications may play a significant role in the pathophysiology of HH.

Clopidogrel is an antiplatelet substance that prevents blood coagulation in the arteries. It is an inactive pro drug that becomes activated after first-pass metabolism by the liver. The active metabolite of clopidogrel is 2-oxoclopidogrel, which is unstable therefore pharmacokinetic data is obtained by measuring the inactive metabolite clopidogrel acid in plasma. Clopidogrel is taken orally in tablet form. The aim of this project was to develop a LC-MS/MS method for quantification of clopidogrel and its metabolite in plasma.   The method has been developed by optimizing the sample preparation. Different extraction procedures and extraction columns were tested, for example, by changing the extraction column from a C8 silica sorbent to Oasis HLB (a polymer sorbent). Different internal standards were evaluated as a result of discovering the signal suppression of the previous internal standard clopidogrel acid.  Flupentixol was found to be the best candidate.