Rozprawy doktorskie na temat „Mass Spectrometric Study”

Abstract This thesis is focused on the hydrolysis of aluminium, the polymerisation of the hydrolysis products, and how these can be monitored by mass spectrometric methods. The main aim of this research is to figure out how the aqueous speciation of aluminium changes as a function of pH (3.2–10), concentration (1–100 mM), reaction time (1s–14d), and counter anion (Cl-, SO42-, HCOO-). The method used was electrospray mass spectrometry. The results showed more variable speciation than those suggested earlier. The main species were Al2, Al3, and Al13, which were found in all of the conditions under scrutiny. The effect of pH was the most remarkable of all the parameters researched. The formation of large highly charged complexes was strongly dependent on it. Also the Al-concentration in the bulk solution had a clear effect on speciation: in dilute solutions there were more protonated ligands and less attached counter anions. This could mean that the species in more diluted bulk solutions had fewer different states of charge. Reaction time caused only minor changes to speciation in the initial pH: there was slightly more variation of a certain sized species in the aged solution. In elevated pH, the birth of important Al13 oligomers was time dependent. The effect of the counter anion was tremendous. In a chloride environment the speciation was rich and diversified. With sulphate the speciation was limited to solid- like compounds, and the variation of single-sized species was almost lacking. The formate as a counter anion caused most surprising results; the charge of aluminium in some studied complexes was lowered from the common 3+ to 1+. If this reaction also occurs in natural circumstances, the uses of aluminium formate would be wide. The results can be utilised in following the progress of dissolution, the mobilization and toxicity of aluminium in natural waters, as well as in water purification, and in reaching minimal chemical contamination levels in sludge as well as in aqueous waste.

Measurements of the signals proportional to the radiation intensities or the number densities of the charged particles in argon ICP plasma are between the most popular methods of analysis of low concentrations of elements. But the relationships between the signals and the quantity of element in the sample are very complicated, depend on many physical phenomena and chemical reactions and must be calibrated by application of the reference materials. The accuracy of analysis depends on the level of agreement between the compositions of the RM matrices and the sample. As the exact matching is not possible the signal formation in the argon plasma and the effects of matrices on the analytical results should be studied. A method of calibration of the dependence of the sensitivity of the mass spectrometer on the ion masses and methods to determine the electron density and temperature from the mass spectrometric measurement data were proposed. Those aid estimation of the ion signals for analysis and assessment of the measurement uncertainty. The largest matrix effects usually are due to easy to ionize elements. The results show that those elements in argon plasma, depending on the ionization potentials and the structure of the atomic energy levels, differ in possibilities to increase the temperature, the temperature and density gradients, deviation from the local thermodynamic equilibrium, take part in charge transfer reaction between doubly ionized ions and argon atoms. The phenomena... [to full text]
Mažos elementų koncentracijos šiuo metu dažniausiai yra nustatomos fizikiniais metodais. Dažnai matuojamas spinduliuotės stipris arba elektringų dalelių skaičius. Tačiau ryšys tarp signalo dydžio ir elemento atomų kiekio yra labai sudėtingas daugelio fizikinių (garavimo ar dulkėjimo, disociacijos, atomų sužadinimo bei jonizacijos, srautų ir kt.) reiškinių bei cheminių reakcijų rezultatas. Jų išeiga kol kas pakankamu tikslumu negali būti numatyta. Todėl tokie matavimai yra tik santykiniai, t.y. jie kalibruojami etaloninėmis medžiagomis. Kalibravimo tikslumas priklauso nuo to kiek bendroji etaloninės medžiagos sudėtis artima tiriamajai medžiagai. Tai įmanoma padaryti tik tam tikru artutinumu. Todėl darbe tiriamos signalo formavimosi sąlygos indukcinėje argono plazmoje ir paskirų paveikiųjų elementų bei bendros bandinio sudėties įtaka bandinių analizinių masių spektrometrinių matavimų rezultatams. Pasiūlytas būdas masių spektrometro jautrio priklausomybei nuo jono masės kalibruoti bei būdai elektronų temperatūrai ir koncentracijai nustatyti iš jonų spektrometrinių matavimų. Tai leidžia įvertinti jonų signalus santykinei pusiaukiekybinei analizei, padeda nustatant matavimų neapibrėžtis. Nustatyta, kad labiausiai analizinį signalą argono plazmos šaltiniuose įtakojantys lengvai jonizuojami elementai, priklausomai nuo jų jonizacijos potencialų ir atominių energijos lygmenų išsidėstymo, gali padidinti plazmos temperatūrą ir gradientus bei nukrypimus nuo dalinės termodinaminės... [toliau žr. visą tekstą]

Resveratrol is a polyphenolic compound produced by various plants and present in dietary sources such as red wine. In recent years, its beneficial effects for human health, including protection from heart diseases and cancer prevention, have attracted increasing interest. Resveratrol acts both as an antioxidant and a prooxidant agent when works in vivo with Cu(II) ions occurring naturally in living organisms. The aim of this work is to study the gas phase reactivity of resveratrol in presence of copper and iron ions, in order to more insights on the role of copper in the proposed biological mechanism. By electrospray ionization (ESI) mass spectrometry we have produced and detected some resveratrol-copper complexes by using a resveratrol/CuSO4 solution in acetonitrile/water, and their most stable structures have been calculated at the B3LYP/6-311G(d) level of theory. The formation of dehydrodimer product was also detected in ESI-MS/MS experiments and its structure assigned with evidences for isomeric compounds from copper and iron reactions with resveratrol. Density Functional Theory (DFT) calculations have been carried out to elucidate reaction mechanisms. Finally, the crucial role of the para-OH group in resveratrol structure has been demonstrated by investigating reactions with copper sulfate of synthetic analogues, bearing different number and position of OH groups.

This study investigates the potential of urinary modified nucleosides as tumour markers. The first task carried out was the development of a reproducible purification method, necessary to extract these compounds from cancer patients' urine, and to separate them from other urinary components that would interfere in further analyses. Secondly the high performance liquid chromatography electrospray ion trap mass spectrometry analytical procedures to be used in the identification and quantification of the modified nucleosides were optimized using commercially available standard compounds. The nucleoside levels in the urine of the healthy population and cancer patients (normalized using urinary creatinine levels) were then compared. The effects of factors other than disease stage (such as age, sex, treatment and site of cancer) upon urinary nucleoside levels were also determined, and the nucleoside profiles of cultured cells, in varying degrees of transformation, and of aseptic fluid from cancer patients examined. Eighteen nucleosides were identified in urine by these procedures. A number of the nucleosides examined exhibited urinary levels that correlated with disease progression. These included N², N^2-dimethylguanosine breast cancer patients and pseudouridine in most of the cancer patients examined. These findings must be considered as exciting but as yet preliminary data from a pilot study, and the potential of these modified nucleosides to act as tumour markers is fully discussed, together with the degree of certainty of the identification of the nucleosides.

Une nouvelle méthode de spectrométrie de masse (TOF MS) à haute température a été développée. La surface de l’échantillon y est chauffée par laser pendant environ 20 ms, et température et spectres de masse sont mesurés en fonction du temps. Chaque expérience couvre tout un intervalle de température. Cette méthode a été appliquée au graphite pyrolytique et au dioxyde d’uranium. L’étude du graphite a clairement montré que la sublimation est de type Langmuir (ou surface libre), malgré les très hautes températures et pressions. Les pressions partielles relatives de C1, C2, C3, C4 et C5 ont été mesurées jusqu’à 4100 K, ainsi que les enthalpies de sublimation des trois espèces principales de la vapeur. Les coefficients d’évaporation relatifs de C1-C5 ont été estimés par comparaison des pressions partielles obtenues ici à 4000 K avec celles à l’équilibre thermodynamique de la littérature. La courbe de pression de vapeur de UO2 au-dessus du dioxyde d’uranium a été mesurée entre 2800 et 3400 K. Des enthalpies de sublimation et de vaporisation sont proposées pour UO2, ainsi qu’une première valeur expérimentale de l’enthalpie de vaporisation de UO3. Les rapports de pressions partielles p(UO2)/p(UO), p(UO2)/p(UO3) et p(UO2+)/p(UO+) ont été mesurés aux alentours de 3300 K et indiquent que l’évaporation se fait dans des conditions proches de l’équilibre thermodynamique. La méthode développée ici est adaptée à l’étude par spectrométrie de masse jusqu’à de très hautes températures de la vaporisation de matériaux réfractaires, et pourrait être utilisée pour l’étude de matériaux chimiquement instables comme le dioxyde d’uranium hyperstœchiométrique ou des carbures et nitrures
A new method of high-temperature mass spectrometry (TOF MS) was developed, where the specimen surface is heated by a laser pulse of approx. 20 ms. During it, time-resolved measurements of mass spectra and of the temperature are performed. Each experiment covers an entire temperature interval. The method was applied to pyrolytic graphite and uranium dioxide. In graphite study, it was clearly shown that the sublimation occurs in a Langmuir-like mode (free surface vaporisation), despite the very high temperatures and thus pressures. Relative partial pressures of C1, C2, C3, C4 and C5 were measured up to 4100 K. Obtained sublimation enthalpies of the main three vapour species are in a good agreement with literature values. Relative vaporisation coefficients of C1-C5 were estimated by comparison of the present partial pressures at 4000 K with equilibrium ones given in the literature. The vapour pressure curve of UO2 over stoichiometric uranium dioxide was measured between 2800 and 3400 K. Obtained sublimation and vaporisation enthalpies are in agreement with the literature. The vaporisation enthalpy of UO3 was measured for the first time. Relative partial pressure ratios p(UO2)/p(UO), p(UO2)/p(UO3) and p(UO2+)/p(UO+) were measured at around 3300 K and indicate that the vaporisation occurs in a regime close to thermodynamic equilibrium. This method is suitable for the fast and time-resolved mass spectrometric measurements of refractory materials up to very high temperatures, and could now be applied to the study of chemically unstable materials such as hyperstoichiometric urania and some carbides and nitrides. Key words: pyrolytic graphite, HOPG, uranium dioxide, laser vaporisation, TOF MS, vaporisation coefficients, Langmuir evaporation

1. Introduction and aim of the work Drug discovery in phytomedicine has in the past been mainly focused on the isolation and characterization of new bioactive compounds from natural products. Several NCE (new chemical entities) have been isolated from plants and they are now the active principles of many drugs able to treat and prevent different kinds of diseases. This drug discovery approach is aimed at the determination of the single "active principle" in plants, based on the assumption that a plant has one or more ingredients which determine its therapeutic effects. Beside NCE derived from plants and herbs, there is another important approach which assumes that a synergy of all ingredients of plants will bring about the maximum of therapeutic efficacy [1]. There are new forms of registered plant-derived medicines (phytomedicines) that are not single chemical entities but a complex mixture of active and inert ingredients derived form a crude extraction. However this approach has long been limited since adequate methods to standardize complex plant mixtures as well as to rationalize complex modes of actions were lacking. Moreover ADMET studies were limited due to the complexity of the phytomedicines. Hence most of the information that is usually retrieved for NCE during the drug discovery stage, such as the ADME profile and the mechanism of action was often not obtained for such complex natural derivatives, limiting their efficacy and application in therapy. Recently, thanks to the advent of novel MS techniques and to the commercial availability of high resolution MS analysers, the opportunity to determine the ADME profiles of plant extracts and to explore their mode of action has become possible. Advanced analytical techniques play an increasingly important role in the characterization, identification and quantification of plant extract compounds, not only in the context of their natural source but also in biological fluids to study their bioavailability and to discover the active compounds. Mass spectrometry has become one of the main standard techniques in this field because of the high sensitivity and specificity of the available mass analyzers. Based on these premises, the aim of my PhD work has been to set-up and apply state of the art MS strategies to better understand the mechanisms of action of some crude plant extracts and in particular tannins and rice extracts as well as to define the absorption and PK profile of cranberry and bilberry standardized extracts which are widely used as therapeutic agents. 2. Set-up and application of MS methods to elucidate biological activities and mechanisms of action of plant extracts During the first part of my Ph.D program, I have used MS strategies to investigate the ability of plant extracts to act as i) sequestering agents of reactive carbonyl species, toxic lipid peroxidation products involved in the pathogenetic mechanisms of several inflammatory based disorders and ii) as protein precipitation agents (tannin effect) using bradykinin, a pro-inflammatory mediator, as protein target. 2.1 Set-up of an isotopic labelling procedure for the characterization of HNE-sequestering agents in natural extracts and its application for the identification of anthocyanidins in black rice giant germ Reactive carbonyl species (RCS) are cytotoxic molecules deriving from the oxidation of sugars and lipids. When the human system undergoes stress condition, the physiological detoxification pathways are not efficient enough to inhibit these reactive molecules. RCS are electrophilic compounds that can easily react with the nucleophilic centers of proteins leading to the generation of Advanced Glycation End Products (AGEs) and Advanced Lipoxidation End Products (ALEs) [2]. These products are involved in many chronic diseases like diabetes, atherosclerosis and neurodegeneration [3]. Plant extracts represent a source of active compounds with a potential RCS quenching ability. The mass spectrometric method we have recently developed [4] was used to screen the ability of water-soluble rice extracts to act as inhibitors of RCS-induced protein carbonylation. Ubiquitin, used as a protein model, was incubated with one of the most reactive RCS, 4-hydroxy-trans-2-nonenal (HNE), and with increasing concentrations of these extracts for 24 h. The amount of modified ubiquitin was determined by HRMS. The inhibitory effect (IC50) of the different extracts was evaluated by measuring the extent of ubiquitin modification in the absence and the presence of the inhibitors. Black rice with giant germ proved to be the most active (IC50 ≈ 29.1 mg/mL). An isotopic signature profile method was applied for the identification of the putative bioactive compounds: the strategy takes advantage of the characteristic isotopic ion cluster produced by the mixture of HNE:2H5-HNE mixed at a 1:1 stoichiometric ratio. The identification of possible bioactive components was obtained by using available databases. Among the list of components identified some anthocyanidins and some aminoacids were present. The capacity of these compounds to act as HNE sequestering agents was tested by HPLC-UV analysis, measuring free HNE not quenched by the molecules tested. The results were expressed as carnosine units. These results support the hypothesis that certain bioactive components sequester RCS and this kind of rice extract can be adopted in dietary strategy for health benefits. 2.2 Development of a direct ESI-MS method to measure the tannin precipitation effect of proline rich peptides Tannins are a heterogeneous class of polyphenols that are present in several plants and foods [5]. Their health benefits, such as antidiarrheal activity [6], are due to their ability to precipitate protein. Thus, it is important to evaluate tannin content in plant extracts to evaluate their potential use as pharmaceutical and nutraceuticals. The purpose of the present work is to set up a suitable method able to quantify the extent of tannin protein precipitation extent. Bradykinin (100 nmoles/mL), chosen as a model, was incubated with increasing concentrations of 1,2,3,4,6-pentagalloyl β-D-glucose and tannic acid chosen as reference of tannic compounds. Bradykinin not precipitated by tannins was determined by a mass spectrometer TSQ Quantum Triple Quadrupole equipped with an ESI source (direct infusion analysis). The results were expressed as PC50 (112.3 µM and 84.6 µM for tannic acid and 1,2,3,4,6-pentagalloyl β-D-glucose respectively). The type of tannin-protein interaction was also evaluated after precipitate solubilization. The involvement of proline residue in tannin-protein interaction was confirmed by repeating the experiment using a synthetized peptide (RR-9) characterized by the same bradykinin sequence, but having proline residues replaced by glycine residues: no interaction occurred between the peptide and tannins. 3. MS methods for the study of the ADME profiles of standardized cranberry and bilberry extracts ADME profile of plant extracts is a quite challenging approach due to the multicomponent nature and chemical complexity of such extracts, their various concentrations across a wide range, the complexity of their interactions and their complex degradation dynamics in vivo. Due to the complexity of both botanicals and biological samples (e.g., blood, urine, tissues), the analytical approaches to monitor the time-dependent concentration profiles of bioavailable plant molecules require a high sensitivity and specificity. The advent of high resolution MS analyzers has fulfilled these requirements, permitting their application in studying ADME profiles of plant extracts. In the second part of my Ph.D thesis I have applied MS techniques to study the ADME profile of cranberry and bilberry extracts in humans and rodents, respectively. I have used both on target and off target MS methods which have permitted a better understanding of the absorption and PK profile of these natural extracts. 3.1 Profiling Vaccinium Macrocarpon components and metabolites in human urine and urines ex-vivo effect on the reduction of C. Albicans adhesion The activity of Cranberry (Vaccinium Macrocarpon) in the prevention of Urinary Tract Infection (UTIs) has long been reported [7]. Controversial results concerning the bioactive compounds are due to the use of different dosages and non-standardized Cranberry extract treatments. In vitro studies reported the activity of PAC-A2 [8], but its involvement as the bioactive molecule is uncertain since it is not detected in human urine [9]. In this work a standardized Cranberry extract (Anthocran™) was administered to four healthy volunteers at a dosage found to be active in human studies (2 capsules/day of Anthocran™, 36 mg proanthocyanidins/capsule) for 7 days. The volunteers followed a diet poor in polyphenols 72 hours before and during the treatment. The urine samples were collected before the beginning of the capsule intake and 1, 2, 4, 6, 10, 12 and 24 hours after the last assumed. An HPLC-MS/MS method was set up for the analyses using an LTQ-XL-Orbitrap mass spectrometer. Two different types of data analyses were performed: on target and off target. The on target data analysis was followed by creating a database containing all the Cranberry extract components and these components were added to other compounds found in literature. The off target strategy consisted of searching all the ions not present or present at an intensity relative to noise in the pre-treatment sample. CFM-ID (competitive fragmentation modeling for metabolite identification) was used for the identification of the unknown ions. These strategies allowed the identification of 35 analytes including Cranberry components, known metabolites and also metabolites hitherto unreported in the literature. Urine collected at different time ranges after the last dosage of Anthocran™ were ex-vivo tested on the reduction of C. Albicans adhesion. Fractions collected after 1 and 12 hours were found effective, significantly reducing adhesion compared to the control (p < 0.001). Purified Anthocran™ components and metabolites identified in the two active urine fractions will be tested. 3.2 Pharmacokinetic profile of bilberry anthocyanins in rats and the role of glucose transporters: LC-MS/MS and computational studies Anthocyanins are pigments widely present in plants and foods and they belong to the flavonoid class. Their health benefits such as antioxidant, anti-inflammatory and anticarcinogenic properties are well reported [10-12], leading to a potential use of these molecules in oxidative-based diseases. Several studies have shown that they are absorbed both in the stomach and in the small intestine [13]. One mechanism of absorption proposed there is the GLUT transporters, a hypothesis supported by an in vitro study on Caco-2 cells [14]. The purpose of this work was to better understand the role of GLUT transporters (GLUT2 and sGLT1) in anthocyanins absorption. A quantitative HPLC-MS/MS method was set up to determine the absorption of 15 anthocyanins present in a standardized bilberry extract (Mirtoselect®, 36% anthocyanins) in rats. The oral treatment was performed under fasted and fed conditions and, in fasting conditions with the co-somministration of glucose. The first group (Group 1) did notundergo any diet or starvation and was treated with Mirtoselect® 100 mg/kg. The second and the third groups were only allowed to drink water 18 h before the treatment and then they were administered respectively with Mirtoselect® 100 mg/kg (Group 2) and Mirtoselect® 100 mg/kg with 1 g/kg glucose (Group 3). The pharmacokinetic parameters were calculated with PK solver add-in program for Microsoft Excel) AUC, TMAX and CMAX. Computational studies were performed to fully understand the molecular recognition of anthocyanins by GLUT2 and sGLT1, to explain the different absorption of anthocyanins found in PK studies and to investigate the electrical form involved in the mechanism of transport. The interaction between anthocyanins and GLUT2 and sGLT1 are quite similar and generally, in the case of anthocyanins, involved: i) H-bonds from all sugar hydroxyl functions, ii) π-π stacking and H-bond from the 5H-chromen-5-one, iii) H-bonds from the 3,4-dihydroxyphenyl moiety. The two simulated transporters yield comparable models, so both proteins might be similarly involved in anthocyanin absorption. The key difference is the greater flexibility of GLUT2 which seems to be able to recognize all the electrical forms tested. 4. Conclusions In conclusion, the present Ph.D work has demonstrated that mass spectrometric strategies based on a high resolution MS analyzer can be successfully applied to better characterize the biological activities and PK profiles of natural extracts characterized by a complex composition. Hence, information usually obtained for pure compounds in the discovery stage can also be retrieved for more complex bioactive products such as plant extracts. Plant extracts which nowadays find a wide use as health products should be better characterized in terms of biological activity and PK thus assuring more efficacy and safety and the fact they are a complex matrix should not limit this information. References [1] Ulrich-Merzenich G, Panek D, Zeitler H, Vetter H, Wagner H, Indian J Exp Biol. 2010, 48: 208-19. [2] Vistoli G., De Maddis D., Cipak A., Zarkovic N., Carini M., Aldini G., Free Radic Res. 2013, 47: 3-27. [3] Nedić O., Rattan S.I., Grune T., Trougakos I.P., Free Radic Res 2013, 47: 28-38. [4] Colzani M., Criscuolo A., De Maddis D., Garzon D., Yeum K.J., Vistoli G., Carini M., Aldini G., J Pharm Biomed Anal. 2014, 91: 108-118. [5] Serrano J., Puupponen-Pimiä R., Dauer A., Aura A.M., Saura-Calixto F., Mol Nutr Food Res. 2009, 53: S310-29. [6] Qin Y., Wang J.B., Kong W.J., Zhao Y.L., Yang H.Y., Dai C.M., Fang F., Zhang L., Li B.C., Jin C., Xiao X.H., J Ethnopharmacol. 2011, 133: 1096-102. [7] Vasileiou, I., Katsargyris A., Theocharis S., Giaginis C., Nutr Res. 2013, 33 : 595-607. [8] Howell, A.B., Vorsa N., Der Marderosian A., Foo L.Y., N Engl J Med. 1998, 339 : 1085-6. [9] Valentova, K., Stejskal D., Bednar P., Vostalova J., Cíhalík C., Vecerova R., Koukalova D., Kolar M., Reichenbach R., Sknouril L., Ulrichova J., Simanek V., J Agric Food Chem. 2007, 55 : 3217-24. [10] Zafra-Stone S., Yasmin T., Bagchi M., Chatterjee A., Vinson J.A., Bagchi D., Mol Nutr Food Res. 2007, 51: 675-83. [11] Joseph S.V., Edirisinghe I., Burton-Freeman B.M., J Agric Food Chem. 2014, 62: 3886-903. [12] Lin B.W., Gong C.C., Song H.F., Cui Y.Y., Br J Pharmacol. 2016,174:1226-1243. [13] He J., Wallace T.C., Keatley K.E., Failla M.L., Giusti M.M., J. Agric. Food Chem., 2009, 7: 3141-3148. [14] Zou T.B., Feng D., Song G., Li H.W., Tang H.W., Ling W.H., Nutrients. 2014,6: 4165-77.

Non enzymatic protein covalent modifications are involved in the toxic effects induced by electrophilic xenobiotics as well as by endogenous cytotoxic oxidation by-products. Aim of my Ph.D work was to set-up MS methods for the identification, characterization and quantification of non-enzymatic covalently modified proteins and peptides in biological matrices. To reach this goal both tandem MS and high resolution approaches were employed due to the wealth of structural and molecular information that these techniques can provide. As a first step the MS methods were applied for understanding in both in vitro and ex vivo conditions the mechanism of protein haptenation induced by amoxicillin (AX). The MS approach was then focused to study in ex vivo condition the covalent reaction between histidine dipeptides, such as carnosine, and toxic endogenous intermediates like reactive carbonyl species (RCS). .

Thesis involves an optimized chromatographic and spectroscopic study of textiles, dyed with natural organic dyestuffs, aiming at the identification of chromophore constituents and their possible degradation products. Liquid chromatography - mass spectrometry coupled with photodiode array detection has been applied for the samples’ analyses after the selective extraction of chromophores from the substrate. The under investigation natural organic dyes are those with a particular cultural value since they have been widely used for textiles staining in the Mediterranean region since Antiquity. Improved “Mild Acid Hydrolysis” with methanol/formic acid enabled the observation of intact organic dyes from the silk textiles, while a water/ACN solvent system has also been employed for the pre treatment of dyestuffs from both reference and silk samples, which has successfully preserved the chemical information of dye compounds in order to create a comprehensive database for the characterization of natural organic dyes in further studies. Moreover, three different chromatographic programs were applied and compared in LC-PDA-MS, and the best separation result from Program B has been employed to the analysis of the real samples from Turkish silk textiles created by a historical dyeing recipes, and a certain amount of effective information in terms of qualitative analysis has been clarified; Περίληψη: Η παρούσα διπλωματική εργασία αφορά την βελτιστοποιημένη χρωματογραφική και φασματοσκοπική μελέτη κλωστοϋφαντουργικών προϊόντων βαμμένων με φυσικές οργανικές χρωστικές, με στόχο την ταυτοποίηση των έγχρωμων συστατικών τους καθώς και πιθανών προϊόντων αποικοδόμησης τους. Οι υπό διερεύνηση φυσικές οργανικές χρωστικές είναι ιδιαίτερης πολιτιστικής αξίας, δεδομένου ότι έχουν χρησιμοποιηθεί ευρέως για την βαφή υφασμάτων στην περιοχή της Μεσογείου από την Αρχαιότητα. Για την εκχύλιση των έγχρωμων ενώσεων των φυσικών οργανικών χρωστικών από τα δείγματα αναφοράς καθώς και από τα ιστορικά δείγματα μεταξιού, εφαρμόστηκε μία ήπια μέθοδος εκχύλισης με σύστημα διαλυτών νερού/ACN σε λουτρό υπερήχων. Επιπλέον, για την απόσπαση των χρωστικών από τα ιστορικά δείγματα προηγήθηκε στάδιο προκατεργασίας με μίγμα μεθανόλης/μυρμηκικού οξέος και θέρμανση σε ήπιες συνθήκες. Η εφαρμογή ήπιων συνθηκών εκχύλισης επέτρεψε την απόσπαση των έγχρωμων ενώσεων των φυσικών οργανικών χρωστικών, διατηρώντας επιτυχώς το σύνολο των χημικών πληροφορίων που θα μας επέτρεπαν τον απόλυτο χαρακτηρισμό των φυσικών χρωστικών. Για την ανάλυση των δειγμάτων μετά την εκλεκτική εκχύλιση των έγχρωμων ενώσεων από τα υποστρώματα, εφαρμόστηκε η υγρή χρωματογραφία – φασματοσκοπία μάζας, LCMS. Συνολικά εφαρμόστηκαν και συγκρίθηκαν τρία διαφορετικά προγράμματα LCMS, εκ των οποίων τα καλύτερα αποτελέσματα διαχωρισμού και ταυτοποίησης επιτεύχθηκαν με το Πρόγραμμα Β. Το πρόγραμμα αυτό εφαρμόστηκε και για την ανάλυση των ιστορικών δειγμάτων από τουρκικά μεταξωτά υφάσματα, βαμμένα βάσει ιστορικών συνταγών βαφής.

El estudio de muestras de pintura persigue diferentes objetivos. Por un lado, identificar los materiales y técnica de la pintura de una época y por lo tanto el know-how de un período histórico. Por otra parte, estudiar la degradación y el envejecimiento de estos materiales con el fin de evitar su desaparición. Al mismo tiempo, se pretende también elegir y aplicar los materiales más adequados en los procesos de restauración y conservación, así como las mejores condiciones de exposición. Desde un punto de vista físico-químico, las pinturas son matrices complejas formadas por capas de una mezcla heterogénea de materiales orgánicos e inorgánicos, mezclados en proporciones desconocidas y dispuestos en capas micrométricas. Además, estos materiales experimentan interacciones entre ellos y un heterogéneo envejecimiento con el tiempo y a exposición a contaminantes, luz y condiciones de conservación, tales como humedad y temperatura. Como resultado, las pinturas no son objetos estáticos, sino sistemas complejos que constituyen una problemática difícil para el químico analítico. La naturaleza de las pinturas requiere, además, de un enfoque de cartografía 2D con el fin de poder, no sólo caracterizar e identificar los materiales presentes, sino también para obtener su distribución en la muestra. El objetivo general de esta tesis es el de rellenar algunas lagunas en el análisis y la caracterización de los sistemas pictóricos. Completar el conocimiento parcial obtenido anteriormente es el hilo conductor, aunque cada capítulo tiene una entidad propia. Con este finalidad se siguieron diferentes caminos contemporáneamente: el desarrollo de métodos de preparación de muestras y los procedimientos analíticos para el estudio de las muestras de pintura, y la caracterización de la composición de los materiales y de sus procesos de degradación mediante la aplicación de las metodologías analíticas desarrolladas. Los diferentes capítulos tratan las siguientes problemáticas: Synchrotron radiation based micro imaging FTIR to the study of painting cross-sections, Characterisation and distribution of oxalates in mural painting samples, MS and SR based techniques for the identification and distribution of painting materials in samples from works of art, GC/MS characterisation of saccharide materials in samples from works-of-art y GC/MS analytical procedure for the characterization of lipids, terpenoidresins, polysaccharide and proteinaceous materials in the presence of interfering inorganic pigments in the same micro sample. La tesis demuestra la capacidad de Radiación Sincrotrón para la obtención de imágenes FTIR en modo de transmisión y la capacidad de Cromatografía de gases/espectrometría de masas para el estudio de los materiales de la pintura y sus productos de degradación, haciendo especial atención a los materiales sacarídicos. La complementariedad de las técnicas ha sido destacada permetiendo una visión global del conjunto.

A new instrument has been designed and constructed to combine matrix isolation infra-red spectroscopy with an electrical discharge supersonic nozzle. The principal aim of this apparatus was to study reactive metal-containing molecules by fragmentation and/or cathode sputtering inside the nozzle. The discharge of chromium and molybdenum hexacarbonyls has been studied by isolating the products in argon matrices. IR spectroscopy was used to identify Cr(CO)n (n = 5,4) and Mo(CO)m (m = 5-3), the first time these metal carbonyls have been produced by discharge fragmentation. The discharge fragmentation of the group 13 metal trialkyls, A1(CH3)3 and Ga(CH3)3 has been investigated. For trimethylaluminium (TMA), which is mainly dimeric at room temperature, the major product was monomeric TMA. No new metal-containing intermediates were identified due to the congested nature of the IR spectra. However, in comparable experiments on trimethylgallium (TMG) which is a monomer under normal conditions, monomethylgallium, GaCH3, has been observed. There have been no previous spectroscopic reports of this molecule. The spectra were assigned with the aid of isotopic substitution studies, as well as ab initio calculations. The final chapter details other tests carried out in the commissioning of the experiment. These have included tests carried out to ascertain the extent of fragmentation in the discharge, the sputtering of metal atoms in the discharge, and the introduction of a time-of-flight spectrometer for rapid optimisation of the discharge conditions.

The characterization of biomolecules and biomolecular complexes represents an area of significant research activity because of the link between structure and function. Drug development relies on structural information in order to target certain domains. Many traditional biochemical techniques, however, are limited by their ability to characterize only certain stable forms of a molecule. As a result, multidimensional approaches, such as ion mobility mass spectrometry coupled to mass spectrometry (IMS-MS), are becoming very attractive tools as they provide fast separation, detection and identification of molecules, in addition to providing three-dimensional shape for structural elucidation. The present work expands the use and application of trapped ion mobility spectrometry-coupled to mass spectrometry (TIMS-MS) by analyzing a range of biomolecules (including proteoforms, intrinsically disordered peptides, DNA and molecular complexes). The aim is to i) evaluate the TIMS platform measuring sensitivity, selectivity, and separation of targeted compounds, ii) pioneer new applications of TIMS for a more efficient and higher throughput methodologies for identification and characterization of biomolecular ions, and iii) characterize the dynamics of selected biomolecules for insight into the folding pathways and the intra-or intermolecular interactions that define their conformational space.

Human serum albumin (HSA) is a plasma protein that fulfils a wide range of biological functions and is thought to be the major Zn2+ transporter in blood plasma. The high affinity Zn2+ binding site (Site A) has recently been characterised and is located at an interdomain site. In addition to metal binding, HSA is also important in the transport of fatty acids. Previous work has shown that the binding of Zn2+ at Site A and the binding of myristate at fatty acid site 2 are mutually exclusive. It has been predicted that upon fatty acid binding, a conformational change occurs that can disrupt the residues that form Site A. This allosteric interaction could have an impact on the Zn2+ dependent activities of histidine-rich glycoprotein (HRG), a plasma protein involved in blood coagulation. The purpose of this work was to investigate the metal-binding properties of HSA and a peptide derived from HRG using a native MS approach. Furthermore, the possible Zn2+ transfer between the proteins was explored and also whether fatty acids influenced the Zn2+ distribution. Native ESI-MS was able to detect Zn2+ ions associating with HSA although the interactions with fatty acids appeared to be broken upon entering the gas phase. No apparent loss of Zn2+ from HSA was observed by ESI-MS following incubation with myristate which was confirmed by elemental analysis in solution. Travelling wave ion mobility-MS showed no significant conformational changes between apo-HSA and holo-HSA although Zn2+ appears to have a role in stabilising the domain I/II interface. HSA incubated with myristate showed a larger collisional cross section that is in agreement with the X-ray crystal structures. A peptide mimicking the His-rich region of HRG, HRGP330, was found to bind up to 5 Zn2+ ions by ESI-MS and evidence from a combination of circular dichroism spectroscopy, ion mobility and top-down MS/MS indicated that a conformational change occurs upon Zn2+ binding. During CID and ETD, Zn2+-binding fragments were able to be detected in order to map which residues Zn2+ was bound to. However, numerous fragments were detected and so it would appear that several possible binding sites in HRGP330 have a similar binding affinity for Zn2+. Complementary ESIMS and elemental analysis showed that up to 90% of Zn2+ was transferred from HSA to HRGP330 even in the absence of fatty acid. Cu2+ also preferentially bound to HRGP330 over the N-terminal peptide mimic of HSA. Overall this could have implications for how these metal ions are transported in blood plasma as it would appear from this evidence that HRG is a significant competitor for metal ions bound to HSA.

Understanding the function and regulation of proteins is critical to medical and physiological research. To achieve this, the modification status, as well as the interactions of proteins need to be defined. Mass spectrometry is often used to catalogue modifications but their effect on interactions is less often reported. In this thesis these two strands are combined to address a series of challenging complexes that underpin cellular processes from stress responses to energy production. Following an introduction to mass spectrometry and its applications to the study of protein interactions (Chapters 1 and 2), three protein assemblies are considered; Hsp70 chaperones, eukaryotic translation initiation factors, and F- and V-type ATP synthases. First, the oligomerisation of Hsp70 is studied (Chapter 3). Dimerisation is shown to be dependent on a highly-conserved phosphorylation site, suggesting a role for this modification in the regulation of protein complex assembly and targeting to antagonistic pathways. A study of eukaryotic translation initiation factor 2B (eIF2B) is presented (Chapter 4), with proteomics revealing clusters of modifications at protein interfaces. Chemical cross-linking is applied, eliminating proposed structural models and supporting a decameric assembly. A cognition-enhancing drug (known as ISRIB) is an inhibitor of the integrated stress response, and was found to stabilise the active decameric eIF2B assembly, rationalising the observed therapeutic effects of ISRIB. Finally, ATP synthases, membrane protein complexes which exhibit multiple forms of regulation, are investigated (Chapter 5). Positional information of small membrane subunits and the effect of post-translational modifications is deduced. The role of an endogenous inhibitor protein is investigated providing insights into its location, oligomeric state, and influence on nucleotide binding. Overall this thesis applies mass spectrometry to three critical protein assemblies and contributes a more complete understanding of their activity by uncovering aspects of their carefully-tuned regulatory mechanisms.

Thesis (M.S.)--West Virginia University, 1999.
Title from document title page. Document formatted into pages; contains viii, 72 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 68-72).

Over the past few years, mass spectrometry-based proteomics has been widely applied to the most diverse fields of biochemistry, biomedicine and biology, and several approaches have been developed to allow absolute and relative quantification of proteins in very complex mixtures. During my PhD, I have conducted three main studies, taking advantage of different quantitative proteomics techniques. In particular SILAC and iTRAQ approaches have been exploited to investigate the calcification process induced by endotoxin in clonal interstitial aortic valve cells, while SILAC and label-free quantitative approaches have been exploited to identify new potential interacting partners and substrates of the protein kinase CK2. Calcific aortic valve disease represents the most common type of valvular disease and the first cause of surgical valve replacement in the industrialized world. No medical therapies are available to prevent or slow down calcium deposition within the valve leaflets, therefore surgery is the only possible treatment. To investigate the molecular mechanisms underlying the calcification process, SILAC and iTRAQ proteomics approaches have been applied to bovine interstitial aortic valve cells, a cellular model that is able to acquire a pro-calcific profile and drive matrix mineralization upon treatment with an inflammatory stimulus like the endotoxin lipopolysaccharide (LPS). The application to the same cellular model of two different quantitative technologies, led to the identification and relative quantification of hundreds of proteins, among which many showed a significant alteration in response to LPS. The acquired data suggest that cellular oxidoreductase activity, cytoskeletal and spliceosome regulation, glycolisis/gluconeogenesis, and arginine metabolism are altered during the acquisition of the pro-calcific profile. These results represent a starting point to investigate more in detail the molecular mechanisms that seem to be strongly involved in the calcification process induced by LPS. The other projects described in this thesis focus on CK2, an essential, constitutively active and highly pleiotropic protein kinase. CK2, like many other kinases, is strongly involved in several cellular processes, and in particular it has been hypothesized that this enzyme plays a crucial role in the transduction of survival signals. However, a clear comprehension of the multiple roles played by this kinase within the cell has not been achieved. The aim of these projects was the identification of interacting partners and substrates of CK2 by means of proteomics approaches to try to shed some light on the functions performed by this kinase. In particular a combination of immunoprecipitation experiments and label-free quantitative analyses has been performed to identify new potential interacting partners of CK2, while the SILAC technology, in combination with the use of a specific and potent inhibitor of CK2, was exploited to identify new putative substrates of this kinase directly in a cellular system. The results obtained confirm the notion that CK2 plays a role in many fundamental cellular functions and clearly indicate a strong involvement of this kinase in the biological processes of protein biosynthesis and degradation. Moreover interesting aspects linked to phosphorylation/dephosphorylation turnover rates emerged from these analyses. A detailed discussion, from a technical and biological point of view, of the data collected is presented. Finally, during my PhD I also collaborated to a project aiming at the identification of the primary molecular targets of antimicrobial photodynamic therapy. This work, not discussed in the thesis, has recently been submitted to the “Journal of Proteomics” with the title: “Molecular Targets of Antimicrobial Photodynamic Therapy Identified by a Proteomic Approach”.
Negli ultimi anni, la ricerca proteomica basata sulla spettrometria di massa è stata applicata in modo esponenziale ai più diversi campi della biochimica, biomedicina e biologia, permettendo il parallelo sviluppo di nuovi approcci per la quantificazione relativa e assoluta delle proteine. Nel corso del mio dottorato, ho seguito lo sviluppo di tre progetti principali, sfruttando diverse tecniche di spettrometria quantitativa. In particolare, le tecnologie SILAC e iTRAQ sono state applicate allo studio del processo di calcificazione delle cellule interstiziali delle valvole aortiche, mentre i metodi SILAC e di quantificazione label-free sono stati sfruttati per l’identificazione di potenziali interattori e substrati della protein chinasi CK2. La calcificazione delle valvole aortiche è una delle più comuni patologie valvolari e prima causa di sostituzione valvolare nei paesi industrializzati. A oggi sfortunatamente non esistono terapie che possano prevenire o curare la deposizione di calcio nelle valvole aortiche, e l’unica soluzione è l’intervento chirurgico. Per chiarire le basi molecolari di questo processo, abbiamo applicato le metodiche SILAC e iTRAQ ad un modello cellulare basato su cellule valvolari cardiache bovine (BVIC), in grado di acquisire un profilo pro-calcifico e favorire la mineralizzazione della matrice extra-cellulare in risposta ad uno stimolo infiammatorio come l’endotossina lipopolisaccaride (LPS). L’utilizzo di due diverse tecnologie allo stesso modello cellulare ha permesso l’identificazione, e la relativa quantificazione, di centinaia di proteine, parecchie delle quali mostrano una significativa alterazione in risposta al trattamento con LPS. L’analisi dei dati ha infatti rivelato l’alterazione di proteine appartenenti a diversi processi cellulari, quali la regolazione del citoscheletro, dei meccanismi ossidoriduttivi e dello spliceosoma, la via metabolica della glicolisi/gluconeogenesi, e il metabolismo dell’arginina, suggerendo il coinvolgimento di queste vie nel fenomeno della calcificazione delle valvole aortiche. Questi risultati rappresentano perciò un punto di partenza per nuovi dettagliati studi dei meccanismi molecolari alla base della calcificazione valvolare indotta da LPS. Gli altri progetti descritti in questa tesi sono focalizzato su CK2, una protein chinasi essenziale, altamente pleiotroica e costitutivamente attiva, fortemente implicata in una moltitudine di processi cellulari, in particolare nella trasduzione dei segnali di sopravvivenza, per la quale sembra giocare un ruolo chiave. Tuttavia una completa comprensione del ruolo che CK2 ricopre nei vari processi cellulari in cui è implicata non è ancora stata raggiunta, perciò questo lavoro ha come scopo l’identificazione di nuovi potenziali interattori e substrati di CK2, allo scopo di chiarire maggiormente la sua funzione all’interno della cellula. Nello specifico, abbiamo abbinato esperimenti d’immunoprecipitazione e analisi quantitativa label-free per lo studio delle proteine che interagiscono con CK2, mentre la tecnologia SILAC combinata con l’uso di un inibitore potente e specifico di CK2 è stata applicata alla ricerca di nuovi potenziali substrati di questa chinasi direttamente in un sistema cellulare. I risultati ottenuti confermano le conoscenze già note riguardo al coinvolgimento di CK2 in diversi processi essenziali per la vita cellulare, e fanno emergere chiaramente un coinvolgimento di primo piano di CK2 nei processi di biosintesi e degradazione proteica. Inoltre, l’analisi dei dati ha anche rivelato interessanti ed inattesi aspetti del turnover di fosforilazione/defosforilazione di proteine fosforilate da CK2. I dati ottenuti sono dettagliatamente presentati in questa tesi, da un punto di vista sia tecnico che biologico. Infine, durante il dottorato ho anche collaborato alla realizzazione di un progetto volto all’identificazione di bersagli molecolari nella terapia fotodinamica antimicrobica, utilizzando un approccio proteomico. Da questa collaborazione, è nato un lavoro (non descritto in questa tesi) che è stato recentemente sottoposto a “Journal of Proteomics” con il titolo: “Molecular Targets of Antimicrobial Photodynamic Therapy Identified by a Proteomic Approach”.

Thesis (Ph.D.)--University of Wollongong, 2005.
Typescript. EMBARGOED-this thesis is subject to a six months embargo (07/09/06) and may only be viewed and copied with the permission of the author. For further information please Contact the Archivist. Includes bibliographical references: leaf 159-194.

Thesis (Ph. D.)--West Virginia University, 2001.
Title from document title page. Document formatted into pages; contains xiii, 165 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 157-165).

The structure elucidation of gas-phase isomeric species $\rm C\sb2H\sb4X\sp+$ (X = F, Cl, Br and I), $\rm C\sb3H\sb6X\sp+$ (X = Cl, and Br), $\rm(C\sb2H\sb5)\sb2O\sp+C\sb2H\sb4X$ (X = Cl and Br), and $\rm C\sb3H\sb6O\sb2\sp{+\cdot}$ has been accomplished by employing tandem mass spectrometric techniques, i.e. metastable ion (MI) mass spectrometry, collision induced dissociation (CID) mass spectrometry, collision induced dissociative ionization (CIDI) mass spectrometry and neutralization reionization (NR) mass spectrometry. For $\rm C\sb2H\sb4X\sp+$ cations, apart from the $\alpha$-isomer, $\rm CH\sb3CHX\sp+,$ the cyclic ethylenehalonium ions, $\rm{\buildrel{{X\sp+}\atop\enspace}\over{CH\sb2\ CH\sb2}}$ are also stable for X = Cl, Br and I (Chapter 3). The two isomers have readily been characterized by CID mass spectrometry and their neutral counterparts have been produced and studied by NR mass spectrometry. It was found that based on an analysis of the heat of formation values of these ions and the electronegativity and polarizibility data of the X atoms, the relative stability of the two isomers is essentially controlled by the C-X bond strength. The relative stability of the cyclic species, however, is also controlled by the polarizibility of the X atom, which indicated that its polarization by the charge-centered carbon involved the outer-electrons in the X atom to form a back-donating bond with the charge-centered carbon. The isomeric halogen substituted triethyloxonium ions $\rm (C\sb2H\sb5)\sb2O\sp+C\sb2H\sb4X,$ (X = Cl and Br) were generated by appropriate gas phase ion molecular reactions between diethylether and appropriate $\rm C\sb2H\sb4XBr\sp{+\cdot}$ via Br$\sp\cdot$ loss (Chapter 4). The $\alpha$-substituted isomer, $\rm (C\sb2H\sb5)\sb2O\sp+CHXCH\sb3,$ and the $\beta$-substituted isomer, $\rm (C\sb2H\sb5)\sb2O\sp+CH\sb2CH\sb2X,$ are both stable in the gas-phase and do not interconvert on a time scale of $10\sp{-5}$s. Three $\rm C\sb3H\sb6X\sp+$ isomers (at least) were found to be stable in the gas-phase (Chapter 5). They are $\rm CH\sb3{-}\sp+CX{-}CH\sb3,\ CH\sb3{-}{\buildrel{{X\sp+}\atop\enspace}\over{CH{-}CH\sb2}},$ and $\rm{\buildrel{CH\sb2-X\sp+\atop\enspace}\over{CH\sb2{-}CH\sb2}}.$ These ions were characterized by their CID mass spectra and their different behavior in forming oxonium ions--$\rm(C\sb2H\sb5)\sb2O\sp+C\sb3H\sb6X$--with diethyl ether. The distonic radical cations $\rm \sp\cdot CH\sb2CH\sb2O\sp+CHOH$ and $\rm \sp\cdot CH\sb2CH\sb2\sp+C(OH)\sb2$, have been directly generated and characterized by their MI and CID mass spectra (Chapter 7). Comparing the dissociative process of $\rm\sp\cdot CH\sb2CH\sb2O\sp+CHOH$ to that of $\rm HCOOCH\sb2CH\sb3\sp{+\cdot}$ led to the conclusion that this distonic ion is the key intermediate in the dissociation of the latter. Thus the previous proposal, based only on the dissociation of HCOOCH$\sb2$CH$\sb3\sp{+\cdot},$ was confirmed. The heat of formation of $\rm\sp\cdot CH\sb2CH\sb2O\sp+CHOH$ was estimated from an appearance energy measurement to be 137 $\pm$ 4 kcalmol$\sp{-1};$ this is 16 kcalmol$\sp{-1}$ lower in energy than HCOOCH$\sb2$CH$\sb3\sp{+\cdot}.$ A detailed study of distonic ions is presented in Chapter 6, including a survey of the common methods to generate and characterize these ions. Properties of such ions which were considered were: stability, isomerization, bond cleavage and ring-strain. The results showed that the characteristics of distonic ions which distinguish them from their conventional counterparts result from the specific interaction of charge and radical sites.

This project investigates the analysis of shallow implants by secondary ion mass spectrometry (SIMS) and the problems that arise from it. Ion implantation is now almost exclusively used in the manufacture of modern very large scale integration devices. The quantification of these implants can be carried out very successfully by SIMS. However the distortions that are present in any SIMS analysis are emphasised when the implanted layer is less than lOOnm below the surface. In order to characterize these distortions, it is necessary to be able to accurately parameterize the implant profile. Taking moments of the data was found to be a reliable method of doing this without constructing a distribution. Once a parameterizing method was found the differential shift was investigated in silicon, with and without a Si-MBE grown capping layer. The results suggested that the differential shift may be a depth dependant phenomenon. The effect of the amorphization of a crystal on ion implantation was investigated with respect to the change in sputter rate going through the damaged region. The effect of uneven etching on this study is discussed in detail. In order to overcome this uneven etching two different raster scan units are discussed. One, is a new totally computer controlled device for use on a new SIMS instrument. The other is a modification to an existing scan unit. This second unit has been used to make craters that are flat to 0.05%.

Atmospheric pressure photoionization (APPI) is an effective ionization technique for the analysis of low polarity and nonpolar compounds using liquid chromatography/mass spectrometry. Ions are produced through a mechanism which begins with initial photoionization of a primary reagent, termed a "dopant", followed by either proton transfer or charge exchange with the analyte(s). This thesis regards improving the ionization efficiency of APPI by identifying new dopant candidates that can increase the breadth of compounds amenable to APPI, and/or improve the ionization efficiency for compounds that are already amenable to APPI. The desired properties for a dopant candidate include high ionization energy (IE) and low reactivity of its photoions with solvent and dopant neutrals. Reactivity tests for 25 substituted-benzene compounds with substituents ranging from strongly electron withdrawing (EW) to strongly electron donating (ED) were performed. Results showed that ED groups decreased reactivity and IE while EW groups increased reactivity and IE; an exception was if the ED group was itself acidic. Of the compounds tested, 2,4-difluoroanisole and 3-(trifluoromethyl)anisole showed the best potential as dopants for charge exchange. These dopants - along with two other novel dopants, bromo- and chlorobenzene - were compared with established dopants (toluene, anisole, and a toluene/anisole mixture) for charge exchange ionization of polycyclic aromatic hydrocarbons (PAHs). Bromo- and chlorobenzene both showed significant improvement in ionization efficiency compared with previously established dopants due to their relatively low reactivity with the solvent and high IE. It was also found that the improved performance for higher IE PAHs, when using anisole, diluted to 0.5% with toluene, was possibly due to the presence of an impurity in anisole. Of the dopants tested, bromobenzene/2,4-difluoroanisole (99.5:0.5 v/v) was determined to be the best overall for charge exchange ionization.

The development of industry, transport, agriculture and power engineering inevitably create problems related to the impact of generated waste on the environment as well as other undesirable processes. The atmosphere is the main component of the Earth’s climate system because changes occurring due to the human economic activity result in serious environmental impacts in all components of the ecosystem as well as the Earth’s climate self-regulation system is disturbed. The objective of this work was to investigate the origin, sources and formation of organic compounds and black carbon in atmospheric aerosol particles and to develop new research methods. To attain this objective the following tasks have been formulated: Development of the identification method for the aerosol origin and source by investigating the carbon isotope mass ratios, illustration of possibilities of the carbon isotope ratio method by identifying the aerosol particle origin during the air mass long-range transport at the Preila Environmental pollution research station, investigation of variation of the black carbon amount in aerosol particles in the diesel engine exhaust depending on the engine working parameters and fuel composition, investigation of the carbon isotope ratio variation in natural aerosol depending on the aerosol particle size distribution, determination of the partial contribution of natural and anthropogenic particles in aerosol by investigation of stable carbon and radiocarbon isotope... [to full text]
Žemės atmosfera yra svarbiausia klimato sferos dalis ir jautriausias antropogeninei taršai aplinkos sandas. Atmosferoje vykstantys procesai pakeičia ekosistemose per šimtmečius nusistovėjusius procesus – stebime įvairias globalinės klimato kaitos sukeltas pasekmes. Mokslinė šio darbo idėja nukreipta į įvairiapusį atmosferos aerozolio dalelių savybių ir prigimties tyrimą bei tyrimo metodų plėtrą, pasiremiant naujausiomis aplinkotyros mokslo žiniomis, akcentuojant pastarojo dešimtmečio masių spektrometrijos pasiekimus. Šio darbo tikslas yra organinių medžiagų ir juodosios anglies atmosferos aerozolio dalelėse kilmės, aerozolio sudėties ir formavimosi tyrimai bei naujų tyrimo metodų plėtra. Darbe eksperimentiškai įrodyta, kad anglies izotopų santykis antropogeninės kilmės aerozolio dalelėse atitinka deginamo kuro izotopų santykį ir šis parametras tinka, identifikuojant aerozolio dalelių šaltinį. Šis izotopų santykis, kaip metodinė priemonė, buvo panaudotas tiriant įvairios prigimties aerozolio sudėtį, savybes ir kilmę. Tiriant aerozolio daleles tolimojoje oro pernašoje stebėjome elementinės anglies δ13C verčių kaitą akumuliacinėje modoje nuo -22,9 ‰ iki -26,3 ‰, organinės anglies δ13C = -28 ‰. Iš δ13C verčių nustatyta, jog elementinės anglies pirmtakai aerozolio dalelėse buvo degimo produktai, organinės anglies šaltinis – lakūs organiniai junginiai iš augalų. Vietinės kilmės dalelių, didesnių už 1 μm, stebėta karbonatinė komponentė. Apjungus masių ir izotopų santykių masių... [toliau žr. visą tekstą]

The measurement of isotopic ratios by inductively coupled plasma mass spectrometry (ICP-MS) has the benefits of ionising all metallic elements, simplifying sample preparation and reducing analysis time, when compared with thermal ionisation mass spectrometry (TIMS). However, the use of ICP-MS in isotopic ratio studies has been somewhat restricted by Its failure to offer the precision and accuracy required by a variety of applications. The precision achievable by ICPMS, typically 0.2 to 0.3 % RSD, for isotopic ratios, has generally been regarded as being primarily limited by instrumental instability. An investigation of the sources of instrumental noise in ICP-MS has been undertaken, utilising noise spectral analysis as a diagnostic md Study of parametric variation upon noise production has identified the methods by which modulation of the ion signal occurs Noise spectral analysis has allowed an understanding of the limitations imposed upon measurement precision by the various contributing noise sources to be established The key to improved measurement precision has been found to lie in the development of data acquisition methods which allow the predominant sources of instrumental noise to be effectively filtered from the ion signal The methodology developed for sequential measurement of isotopes, using a quadrupole mass analyser, to reduce the deleterious influences of instrumental noise is discussed. Results are given for isotopic ratio measurement which demonstrate that a precision of approximately 0 05 % RSD can be attained The factors which affect the accuracy of isotopic ratio measurement are shown to be many and varied and depend to a large extent on the particular Isotopes bemg studied Definition of an appropriate measurement strategy for high accuracy isotope ratio measurement involves consideration of all possible causes of bias and adoption of methods for their elimination or correction. To facilitate this process a protocol has been developed and subsequently applied to various elements and instrument systems.

Respiratory Distress Syndrome (RDS) is a respiratory disorder that can affect preterm newborns. It is due to lung immaturity and a deficiency of lung surfactant, a thin lipoprotein layer which allows lung expansion and prevents alveolar collapse during expiration. In these babies, nasal Continuous Positive Airway Pressure (nCPAP) provides non-invasive respiratory support by alveolar recruitment and improves functional residual capacity during spontaneous breathing. In case nCPAP fails, the next step is treatment with exogenous surfactant, which improves gas exchange and survival, reducing the need for mechanical ventilation and then the incidence of chronic lung disease. Although surfactant replacement therapy remains the gold standard for the treatment of RDS, failure rate ranges from 9% to 50%. The possibility of tracing the fate of the administered drug and estimate in vivo the amount of drug reaching the lung could help in improving the therapy and allow the premise to test different delivery systems. RDS is a disorder related to prematurity. Among the factors responsible for premature birth, intrauterine infection (chorioamnionitis) is one of the main causes and it has been reported to have effects on the development of fetal organs. In particular, it has been reported that infants exposed to chorioamnionitis had a reduced risk to develop RDS but an increased risk for chronic lung diseases. Since the function of lung surfactant strictly depends on its composition, a better knowledge on surfactant metabolite and lipid changes would elucidate the effects of the exposition of the foetus to maternal intrauterine infection on RDS. The PhD project focused on 2 objectives: 1. To validate natural abundance stable isotopes approach as a reliable method to quantify the contribution of exogenous surfactant to the alveolar surfactant pool in a rabbit model of RDS treated with a porcine exogenous surfactant; 2. To assess the effect of maternal intrauterine infection on the surfactant composition of newborns affected by RDS by liquid chromatography-mass spectrometry (LC-MS/MS) based metabolomics .

ESI-MS screening methods directly detect ligand-target non covalent complexes in the gas phase and allow inference of affinity (and specificity) of the ligand-target interaction in solution [1, 2]. The identity of different complexes can be directly assessed as the mass of each molecule works as intrinsic label. Biopolymers can be screened either as a single component or a mixture of different targets; in this way it is possible to determine the selectivity of a new chemical entity for different targets. On the other hand, using ESI-MS it is also possible to identify, within a mixture, components that selectively bind the active site of a certain biopolymer and could be profitably used to screen libraries of known compounds. The aim of this PhD project was the study by ESI mass spectrometry of different noncovalent complexes formed with biopolymers identified as possible therapeutic targets (Mismatch Repair Mechanism and Hsp90). The noncovalent interaction between biological targets and possible ligands were studied in order to identify potential inhibitors. The binding affinities determined by ESI-MS were compared to existing data obtained by solution phase methods. A novel MS-based method was implemented for testing different biopolymer, of therapeutic interest, against a library of fragments (molecular weight 100-300 Da) constituted of about 2,000 compounds. Particularly this approach was used for the identification of new molecules able to recognize TG mismatched base pairs in DNA that are responsible for most of the common mutations leading to formation of tumors in humans. TG mismatches are particularly abundant in cells lacking mismatch repair mechanism (MMR). MMR is involved in the correction of DNA polymerase errors that escape proofreading activity. MMR deficiency increases 50-1000-fold spontaneous mutation rates (microsatellite instability MSI) and in addition, MMR deficiency can lead to resistance to several chemotherapeutic agents (DNA damaging agent) [3]. An ESI-MS method was used to study the complexes formed between different DNA duplexes and minor groove binders and intercalator compounds. A hairpin DNA sequence (CTGGsm) bearing a single T:G mismatch and a matched hairpin DNA sequence (CCGG) were prepared and used to set up the method. Two DNA sequences were also synthesized: a self complementary G:C rich DNA sequence (HFM) and a polyAT DNA duplex (A5TG). The association constants (KAs) were directly determined from the MS spectrum and the amount of bound ligand was used to determine the selectivity of a binder among the different DNA sequences. Minor groove binders confirmed their selectivity toward AT rich DNA duplex (A5TG) whereas a tris imidazole lexitropsin derivative proved to be selective for the TG mismatched DNA hairpin (CTGGsm) in agreement with NMR, SPR, and ITC studies. A medium throughput screening (MTS) method was setup for the screening of the fragment library (about 1,000 cps) and the procedure was validated studying the interactions between the two different DNA sequences (HFM and CTGGsm) and minor groove binders. The screening was performed on the fragment library and hit compounds were afterwards tested against HFM and CCGG in order to identify selective ligands. Among the different hits identified, a slight selectivity (1.3) for the single mismatched sequence was highlighted for the fragment FBA-05-094. Twenty close analogues of FBA-05-094 were subsequently tested against CTGGsm and CCGG DNA with the aim to find more selective and higher affinity ligands. The screening of the fragment library on CTGGsm was repeated using ligand mixtures (5 and 10 components) in order to improve the assay throughput. A good agreement between the results obtained by screening the compounds as single component and in mixture was found. We investigated also the binding of small molecules towards two enzymes (PKA and Hsp90) in order to verify the possibility to apply this procedure to protein targets. Hsp90 (heat shock protein 90) is a molecular chaperone and is one of the most abundant proteins expressed in cell [4]. Targeting Hsp90 with drugs has shown promising effects in clinical trials. Inhibition of the Hsp90 ATPase machinery by natural, semi- and totally synthetic inhibitors has shown promising results in clinic. The interactions between Hsp90 and different known ligands that bind either at N or C terminal binding domains were studied and the dissociation constants for this set of ligands (KD from 0.0007 uM to 103 uM) were previously determined by fluorescence polarization (FP). We found a good agreement between these values and the percentage of bound protein determined by mass spectrometry. A screening of the fragment library against Hsp90 was performed and the resulted hit compounds were compared to those obtained by using NMR FAXS technique[5]. A good correlation was found between the data obtained by MS and NMR screenings. Competition experiments using a known ATP competitor were performed and these studies highlighted the presence of a few ATP competitor ligands in agreement with X-ray experiments. [1] Ganem B, Li Y.-T., Henion JD. Detection of noncovalent Receptor-Ligand complexes by Mass Spectrometry. Journal of American Society for Mass Spectrometry, 113, 6294-6296 (1991) [2] Hofstadler SA, Sannes-Lowery KA. Applications of ESI-MS in drug discovery: interrogation of noncovalent complexes. Nature Reviews Drug Discovery, 5(7), 585-595 (2006) [3] Stojic L., Brun R., Jiricny J., Mismatch repair and DNA damage signalling. DNA Repair, 3, 1091-1101 (2004). [4] Chaudhury S., Welch T.R., Blagg B.S., Hsp90 as a Target for Drug Development. ChemMedChem, 1331-1340 (2006). [5] Dalvit C., Fagerness P.E., Hadden D.T.A., Sarver R.W., Stockman B.J. Fluorine-NMR Experiments for High-Throughput Screening: Theoretical Aspects, Practical Considerations, and Range of Applicability. Journal of American Society for Mass Spectrometry, 125, 7696-7703 (2003)