Drug Analysis without Primary Reference Standards : Application of LC-TOFMS and LC-CLND to Biofluids and Seized Material

Drug Analysis without Primary Reference Standards: Application of LC-TOFMS and LC-CLND to Biofluids and Seized Material Primary reference standards for new drugs, metabolites, designer drugs or rare substances may not be obtainable within a reasonable period of time or their availability may also be hindered by extensive administrative requirements. Standards are usually costly and may have a limited shelf life. Finally, many compounds are not available commercially and sometimes not at all. A new approach within forensic and clinical drug analysis involves substance identification based on accurate mass measurement by liquid chromatography coupled with time-of-flight mass spectrometry (LC-TOFMS) and quantification by LC coupled with chemiluminescence nitrogen detection (LC-CLND) possessing equimolar response to nitrogen. Formula-based identification relies on the fact that the accurate mass of an ion from a chemical compound corresponds to the elemental composition of that compound. Single-calibrant nitrogen based quantification is feasible with a nitrogen-specific detector since approximately 90% of drugs contain nitrogen. A method was developed for toxicological drug screening in 1 ml urine samples by LC-TOFMS. A large target database of exact monoisotopic masses was constructed, representing the elemental formulae of reference drugs and their metabolites. Identification was based on matching the sample component s measured parameters with those in the database, including accurate mass and retention time, if available. In addition, an algorithm for isotopic pattern match (SigmaFit) was applied. Differences in ion abundance in urine extracts did not affect the mass accuracy or the SigmaFit values. For routine screening practice, a mass tolerance of 10 ppm and a SigmaFit tolerance of 0.03 were established. Seized street drug samples were analysed instantly by LC-TOFMS and LC-CLND, using a dilute and shoot approach. In the quantitative analysis of amphetamine, heroin and cocaine findings, the mean relative difference between the results of LC-CLND and the reference methods was only 11%. In blood specimens, liquid-liquid extraction recoveries for basic lipophilic drugs were first established and the validity of the generic extraction recovery-corrected single-calibrant LC-CLND was then verified with proficiency test samples. The mean accuracy was 24% and 17% for plasma and whole blood samples, respectively, all results falling within the confidence range of the reference concentrations. Further, metabolic ratios for the opioid drug tramadol were determined in a pharmacogenetic study setting. Extraction recovery estimation, based on model compounds with similar physicochemical characteristics, produced clinically feasible results without reference standards.

Impact of low temperature storage on active and storage forms of folate in choy sum (Brassica rapa subsp parachinensis)

Choy sum (Brassica rapa subsp. parachinensis) is a dark green leafy vegetable that contains high folate (vitamin B9) levels comparable to spinach. Folate is essential for the maintenance of human health and is obtained solely through dietary means. Analysis of the edible portion of choy sum by both microbiological assay and LC-MS/MS indicated that total folate activity remained significantly unchanged over 3 weeks storage at 4 degrees C. Inedible fractions consisted primarily of outer leaves, which showed signs of rotting after 14d, and a combination of rotting and yellowing after 21 d, contributing to 20% and 40% of product removal, respectively. Following deconjugation of the folate present in choy sum to monoglutamate and diglutamate derivatives, the principal forms (vitamers) of folate detected in choy sum were 5-methyltetrahydrofolate and 5-formyl tetrahydrofolate, followed by tetrahydrofolate (THF), 5,10-methenyl-THF, and 10-formyl folic acid. During storage, a significant decline in 5-formyl-THF was observed, with a slight but not significant increase in the combined 5-methyl-THF derivatives. The decline in 5-formyl-THF in relation to the other folate vitamers present may indicate that 5-formyl-THF is being utilised as a folate storage reserve, being interconverted to more metabolically active forms of folate, such as 5-methyl-THF. Although folate vitamer profile changed over the storage period, total folate activity did not significantly change. From a human nutritional perspective this is important, as while particular folate vitamers (e.g. 5-methyl-THF) are necessary for maintaining vital aspects of plant metabolism, it is less important to the human diet, as humans can absorb and interconvert multiple forms of folate. The current trial indicates that it is possible to store choy sum for up to 3 weeks at 4 degrees C without significantly affecting total folate concentration of the edible portion. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.

HPLC analysis of plant sterol oxidation products

Increased interest in the cholesterol-lowering effect of plant sterols has led to development of plant sterol-enriched foods. When products are enriched, the safety of the added components must be evaluated. In the case of plant sterols, oxidation is the reaction of main concern. In vitro studies have indicated that cholesterol oxides may have harmful effects. Due their structural similarity, plant sterol oxidation products may have similar health implications. This study concentrated on developing high-performance liquid chromatography (HPLC) methods that enable the investigation of formation of both primary and secondary oxidation products and thus can be used for oxidation mechanism studies of plant sterols. The applicability of the methods for following the oxidation reactions of plant sterols was evaluated by using oxidized stigmasterol and sterol mixture as model samples. An HPLC method with ultraviolet and fluorescence detection (HPLC-UV-FL) was developed. It allowed the specific detection of hydroperoxides with FL detection after post-column reagent addition. The formation of primary and secondary oxidation products and amount of unoxidized sterol could be followed by using UV detection. With the HPLC-UV-FL method, separation between oxides was essential and oxides of only one plant sterol could be quantified in one run. Quantification with UV can lead to inaccuracy of the results since the number of double bonds had effect on the UV absorbance. In the case of liquid chromatography-mass spectrometry (LC-MS), separation of oxides with different functionalities was important because some oxides of the same sterol have similar molecular weight and moreover epimers have similar fragmentation behaviour. On the other hand, coelution of different plant sterol oxides with the same functional group was acceptable since they differ in molecular weights. Results revealed that all studied plant sterols and cholesterol seem to have similar fragmentation behaviour, with only relative ion abundances being slightly different. The major advantage of MS detection coupled with LC separation is the capability to analyse totally or partly coeluting analytes if these have different molecular weights. The HPLC-UV-FL and LC-MS methods were demonstrated to be suitable for studying the photo-oxidation and thermo-oxidation reactions of plant sterols. The HPLC-UV-FL method was able to show different formation rates of hydroperoxides during photo-oxidation. The method also confirmed that plant sterols have similar photo-oxidation behaviour to cholesterol. When thermo-oxidation of plant sterols was investigated by HPLC-UV-FL and LC-MS, the results revealed that the formation and decomposition of individual hydroperoxides and secondary oxidation products could be studied. The methods used revealed that all of the plant sterols had similar thermo-oxidation behaviour when compared with each other, and the predominant reactions and oxidation rates were temperature dependent. Overall, these findings showed that with these LC methods the oxidation mechanisms of plant sterols can be examined in detail, including the formation and degradation of individual hydroperoxides and secondary oxidation products, with less sample pretreatment and without derivatization.

Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.

Enrichment and identification of glycoproteins in human saliva using lectin magnetic bead arrays

Aberrant glycosylation of proteins is a hallmark of tumorigenesis, and could provide diagnostic value in cancer detection. Human saliva is an ideal source of glycoproteins due to the relatively high proportion of glycosylated proteins in the salivary proteome. Moreover, saliva collection is non-invasive, technically straightforward and the sample collection and storage is relatively easy. Although, differential glycosylation of proteins can be indicative of disease states, identification of differential glycosylation from clinical samples is not trivial. To facilitate salivary glycoprotein biomarker discovery, we optimised a method for differential glycoprotein enrichment from human saliva based on lectin magnetic bead arrays (saLeMBA). Selected lectins from distinct reactivity groups were used in the saLeMBA platform to enrich salivary glycoproteins from healthy volunteer saliva. The technical reproducibility of saLeMBA was analysed with LC-MS/MS to identify the glycosylated proteins enriched by each lectin. Our saLeMBA platform enabled robust glycoprotein enrichment in a glycoprotein- and lectin-specific manner consistent with known protein-specific glycan profiles. We demonstrated that saLeMBA is a reliable method to enrich and detect glycoproteins present in human saliva.

Monitoring temporal changes in use of two cathinones in a large urban catchment in Queensland, Australia

Wastewater analysis was used to examine prevalence and temporal trends in the use of two cathinones, methylone and mephedrone, in an urban population (>200,000 people) in South East Queensland, Australia. Wastewater samples were collected from the inlet of the sewage treatment plant that serviced the catchment from 2011 to 2013. Liquid chromatography coupled with tandem mass spectrometry was used to measure mephedrone and methylone in wastewater sample using direct injection mode. Mephedrone was not detected in any samples while methylone was detected in 45% of the samples. Daily mass loads of methylone were normalized to the population and used to evaluate methylone use in the catchment. Methylone mass loads peaked in 2012 but there was no clear temporal trend over the monitoring period. The prevalence of methylone use in the catchment was associated with the use of MDMA, the more popular analogue of methylone, as indicated by other complementary sources. Methylone use was stable in the study catchment during the monitoring period whereas mephedrone use has been declining after its peak in 2010. More research is needed on the pharmacokinetics of emerging illicit drugs to improve the applicability of wastewater analysis in monitoring their use in the population.

Proteomic identification of putative microRNA394 target genes in Arabidopsis thaliana identifies MLP family members critical for normal development

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem (SAM) organisation. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Major Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR. Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development.

Mass spectrometry and n-in-one analytics in early drug discovery: Combinatorial chemistry libraries, lipophilicity and absorption screening

This thesis describes current and past n-in-one methods and presents three early experimental studies using mass spectrometry and the triple quadrupole instrument on the application of n-in-one in drug discovery. N-in-one strategy pools and mix samples in drug discovery prior to measurement or analysis. This allows the most promising compounds to be rapidly identified and then analysed. Nowadays properties of drugs are characterised earlier and in parallel with pharmacological efficacy. Studies presented here use in vitro methods as caco-2 cells and immobilized artificial membrane chromatography for drug absorption and lipophilicity measurements. The high sensitivity and selectivity of liquid chromatography mass spectrometry are especially important for new analytical methods using n-in-one. In the first study, the fragmentation patterns of ten nitrophenoxy benzoate compounds, serial homology, were characterised and the presence of the compounds was determined in a combinatorial library. The influence of one or two nitro substituents and the alkyl chain length of methyl to pentyl on collision-induced fragmentation was studied, and interesting structurefragmentation relationships were detected. Two nitro group compounds increased fragmentation compared to one nitro group, whereas less fragmentation was noted in molecules with a longer alkyl chain. The most abundant product ions were nitrophenoxy ions, which were also tested in the precursor ion screening of the combinatorial library. In the second study, the immobilized artificial membrane chromatographic method was transferred from ultraviolet detection to mass spectrometric analysis and a new method was developed. Mass spectra were scanned and the chromatographic retention of compounds was analysed using extract ion chromatograms. When changing detectors and buffers and including n-in-one in the method, the results showed good correlation. Finally, the results demonstrated that mass spectrometric detection with gradient elution can provide a rapid and convenient n-in-one method for ranking the lipophilic properties of several structurally diverse compounds simultaneously. In the final study, a new method was developed for caco-2 samples. Compounds were separated by liquid chromatography and quantified by selected reaction monitoring using mass spectrometry. This method was used for caco-2 samples, where absorption of ten chemically and physiologically different compounds was screened using both single and nin- one approaches. These three studies used mass spectrometry for compound identification, method transfer and quantitation in the area of mixture analysis. Different mass spectrometric scanning modes for the triple quadrupole instrument were used in each method. Early drug discovery with n-in-one is area where mass spectrometric analysis, its possibilities and proper use, is especially important.

Valittujen makrolidien ominaisuudet ja niiden vaikutukset fermentoinnin jälkeiseen jatkokäsittelyyn

Työn kirjallisessa osuudessa tarkasteltiin makrolideja yleisellä tasolla keskittyen kahden makrolidin ominaisuuksiin molekyylitasolla. Näiden tautomerisoitumista käsitellään tuoden esiin sekä yhdisteiden rakenteelliset yhteneväisyydet ja eroavaisuudet että niiden vaikutukset yhdisteiden vaikutusmekanismiin ja metaboliaan. Kirjallisessa osuudessa perehdyttiin myös makrolidien biosynteesiin ja tuotantoprosessiin keskittyen downstream-prosessointiin ja erityisesti biosynteesistä peräisin oleviin epäpuhtauksiin. Lisäksi kirjallisessa osuudessa käsiteltiin argentaatiokromatografian perusteita. Kokeellisessa osuudessa yhdelle makrolidille kehitettiin argentaatiokromatografiaan perustuva puhdistusmenetelmä. Perinteisillä kromatografisilla menetelmillä yhdistettä ei voida puhdistaa kaikista sen epäpuhtauksista. Makrolidin puhtaus argentaatiokromatografian jälkeen oli 98,6 %. Lisäksi kehitettiin uusi kiteytysmenetlmä, jossa yhdiste kiteytettiin anhydridina tavanomaisen monohydraattimuodon sijasta. Makrolidin analysointiin kehitettiin HPLC-menetelmä, joka validoitiin ICH:n ohjeiden mukaisesti. Yhditeen tautomeerimuodot ja siinä esiintyvät epäpuhtaudet tutkittiin käyttäen LC/MS-analyysia. Yhden epäpuhtauden rakenne varmistettiin eristämisen jälkeen NMR:n avulla. Saatavilla olevien tietojen mukaan yhdisteen tulkittuja NMRspektrejä ei ole julkaistu. Lisäksi aiemmin tuntematon epäpuhtaus identifioitiin perustuen retentioaikaan ja MS-analyysiin.

Proteomics of Trypanosoma evansi Infection in Rodents

Background: Trypanosoma evansi infections, commonly called 'surra', cause significant economic losses to livestock industry. While this infection is mainly restricted to large animals such as camels, donkeys and equines, recent reports indicate their ability to infect humans. There are no World Animal Health Organization (WAHO) prescribed diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable and very little efforts are being made to develop improved methods of prevention, diagnosis and treatment. With a view to identify potential diagnostic markers and drug targets we have studied the clinical proteome of T. evansi infection using mass spectrometry (MS).Methodology/Principal Findings: Using shot-gun proteomic approach involving nano-lc Quadrupole Time Of Flight (QTOF) mass spectrometry we have identified over 160 proteins expressed by T. evansi in mice infected with camel isolate. Homology driven searches for protein identification from MS/MS data led to most of the matches arising from related Trypanosoma species. Proteins identified belonged to various functional categories including metabolic enzymes; DNA metabolism; transcription; translation as well as cell-cell communication and signal transduction. TCA cycle enzymes were strikingly missing, possibly suggesting their low abundances. The clinical proteome revealed the presence of known and potential drug targets such as oligopeptidases, kinases, cysteine proteases and more.Conclusions/Significance: Previous proteomic studies on Trypanosomal infections, including human parasites T. brucei and T. cruzi, have been carried out from lab grown cultures. For T. evansi infection this is indeed the first ever proteomic study reported thus far. In addition to providing a glimpse into the biology of this neglected disease, our study is the first step towards identification of diagnostic biomarkers, novel drug targets as well as potential vaccine candidates to fight against T. evansi infections.

A Curious Harpour in Helle : An Edition of the Commentary on the Orpheus Metre of De consolatione philosophiae in MS Thott 304,2

Tutkielmani on tekstieditio keskiaikaisesta kommentaarista, joka löytyy Kööpenhaminan Kuninkaallisen kirjaston käsikirjoituksesta Thott 304,2. Käsikirjoitus voidaan ajoittaa 1400-luvun ensimmäiselle neljännekselle ja se on kirjoitettu keskienglanniksi. Se sisältää John Waltonin runomuotoisen käännöksen Boethiuksen 500-luvun alussa latinaksi kirjoittamasta teoksesta De consolatione philosophiae (Filosofian lohdutus) sekä teosta kommentoivan proosamuotoisen kommentaarin. Käsikirjoitus on vaillinainen, mutta sen säilyneet foliot ovat enimmäkseen erittäin hyväkuntoisia. Itse käsikirjoitusta on tutkittu vain muutamissa artikkeleissa ja yhdessä tutkielmassa; kommentaarista ei ole toistaiseksi tehty kattavaa tutkimusta. Tavoitteeni onkin tuoda kommentaari keskiaikaisen filosofian, keskienglannin ja käsikirjoitusten tutkijoiden käytettäviin. Käsikirjoitus Thott 304,2 on esimerkki epätyypillisestä myöhäiskeskiaikaisesta maallikkomesenaattiudesta Englannissa. Tavanmukaisesti maallikkomesenaatit tukivat uskonnollisten tekstien tuottamista ja kääntämistä, kun taas Thott 304,2 sisältää käännöksen filosofisesta tekstistä. Sen lisäksi mesenaatti oli nainen, aatelistoon kuuluva Elizabeth Berkeley. Varsin harvinaiseksi käsikirjoituksen tekee sen painaminen kirjaksi 1500-luvun alussa. Kirjanpainajan käsikirjoituksen sivuille tekemistä merkinnöistä saadaan korvaamatonta tietoa varhaisesta painotekniikasta, sillä kirjanpainajien käyttämiä käsikirjoituskopioita ei ole säilynyt kovin runsaasti. Waltonin käännös on säilynyt yli kahdessakymmenessä käsikirjoituskopiossa, joista vain Thott 304,2 sisältää laajan kommentaarin. 1500-luvun painoksesta on jäljellä kolme kopiota, ja ne sisältävät saman kommentaarin kuin Thott 304,2. Valitsin editoitavaksi niin kutsuttua Orfeus-runoa kommentoivan osan kommentaarista, sillä se muodostaa ehjän kokonaisuuden ja sopii pituutensa puolesta Pro gradu -tutkielmaan. Orfeus-runo on myös yksi käsikirjoituksen kattavimmin kommentoiduista runoista. Editio on niin sanottu diplomaattinen transkriptio, jossa käsikirjoituksen piirteet on pyritty säilyttämään mahdollisimman tarkasti edition luettavuuden siitä kuitenkaan kärsimättä. Perinteisistä editioista poiketen tutkielmani sisältää myös kommentaarin ja Orfeus-runon transkriptiot, joissa rivijako, lyhenteet ja erikoismerkit on säilytetty. Näiden transkriptioiden toivon auttavan erityisesti käsikirjoituksessa esiintyvien lyhenteiden, erikoismerkkien ja kirjanpainajan merkintöjen tulkinnassa ja tutkimisessa. Editiota ja transkriptioita täydentävät nykyenglanniksi kirjoitettu lyhennelmä kommentaarista ja kuvat käsikirjoituksen sivuista, joilla editoimani kommentaari on. Tutkielmaan sisältyy alkuperäisen tekstin, käännöksen, kommentaarin ja käsikirjoituksen taustaa valottava osuus. Esittelen myös kaikki löytämäni lähteet, joissa käsikirjoitus on mainittu tai joissa sitä on tutkittu. Liitteeksi olen laatinut sanaston helpottamaan kommentaarin tulkitsemista.

Method-MS, final report 2010

Radiometric determination methods, such as alpha spectrometry require long counting times when low activities are to be determined. Mass spectrometric techniques as Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Thermal Ionisation Mass Spectrometry (TIMS) and Accelerator Mass Spectrometry (AMS) have shown several advantages compared to traditional methods when measuring long-lived radionuclides. Mass spectrometric methods for determination of very low concentrations of elemental isotopes, and thereby isotopic ratios, have been developed using a variety of ion sources. Although primarily applied to the determination of the lighter stable element isotopes and radioactive isotopes in geological studies, the techniques can equally well be applied to the measurement of activity concentrations of long-lived low-level radionuclides in various samples using “isotope dilution” methods such as those applied in inductively coupled plasma mass spectrometry (ICP-MS). Due to the low specific activity of long-lived radionuclides, many of these are more conveniently detected using mass spectrometric techniques. Mass spectrometry also enables the individual determination of Pu-239 and Pu-240, which cannot be obtained by alpha spectrometry. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) are rapidly growing techniques for the ultra-trace analytical determination of stable and long-lived isotopes and have a wide potential within environmental science, including ecosystem tracers and radio ecological studies. Such instrumentation, of course needs good radiochemical separation, to give best performance. The objectives of the project is to identify current needs and problems within low-level determination of long-lived radioisotopes by ICP-MS, to perform intercalibration and development and improvement of ICP-MS methods for the measurement of radionuclides and isotope ratios and to develop new methods based on modified separation chemistry applied to new auxiliary equipment.

Identifying N60D mutation in omega subunit of Escherichia coli RNA polymerase by bottom-up proteomic approach

Escherichia coli RNA polymerase is a multi-subunit enzyme containing alpha(2)beta beta'omega sigma, which transcribes DNA template to intermediate RNA product in a sequence specific manner. Although most of the subunits are essential for its function, the smallest subunit omega (average molecular mass similar to 10,105 Da) can be deleted without affecting bacterial growth. Creating a mutant of the omega subunit can aid in improving the understanding of its role. Sequencing of rpoZ gene that codes for omega subunit from a mutant variant suggested a substitution mutation at position 60 of the protein: asparagine (N) -> aspartic acid (D). This mutation was verified at the protein level by following a typical mass spectrometry (MS) based bottom-up proteomic approach. Characterization of in-gel trypsin digested samples by reverse phase liquid chromatography (LC) coupled to electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) enabled in ascertaining this mutation. Electron transfer dissociation (ETD) of triply charged (M + 3H)(3+)] tryptic peptides (residues 53-67]), EIEEGLINNQILDVR from wild-type and EIEEGLIDNQILDVR from mutant, facilitated in unambiguously determining the site of mutation at residue 60.

A metabolomic approach to studying mechanisms of polymeric gene delivery to retinal pigment epithelial cells

Tiivistelmä ReferatAbstract Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.