Direct open air surface sampling/ionization mass spectrometry methods in the study of neutral and nonpolar compounds

Direct open air surface sampling/ionization mass spectrometry offers a means to the rapid, sensitive, and reliable analysis of samples in their native states. It can be used for fingerprinting, but also for studying the distribution of compounds on surfaces by mass spectrometry imaging (MSI). The aim of this work was to develop and apply direct open air surface sampling/ionization mass spectrometry methods in the study of neutral and nonpolar compounds, with emphasis on photoionization-based techniques. First, two krypton discharge photoionization lamps were compared in micro atmospheric pressure photoionization and desorption atmospheric pressure photoionization mass spectrometry (DAPPI-MS). The radio frequency (rf) alternating current lamp was shown to have even over an order of magnitude higher ionization efficiency than the direct current (dc) lamp, thus providing better sensitivity. This was possibly because the rf lamp emitted 5 times the number of photons of the dc lamp. Next, a polydimethylsiloxane (PDMS) extraction phase was developed and optimized for DAPPI-MS analyses of aqueous samples to decrease the severe matrix effects often encountered in direct open air surface sampling/ionization mass spectrometry. The method was applied in the analysis of spiked wastewater and urine samples. The direct open air surface sampling/ionization mass spectrometry methods best suited for neutral and nonpolar analytes do not enable analyses at sub-millimeter range, and thus they are not optimal for MSI. A novel laser ablation atmospheric pressure photoionization (LAAPPI) ion source was developed to enable analyses of at ~300 μm resolution. The LAAPPI method was capable of semi-quantitative performance and suited for the analysis of neutral and nonpolar compounds directly from plant and animal tissues. Subsequent work also demonstrated the suitability of LAAPPI for MSI with sage (Salvia officinalis) leaves as tissue model. The final part of the research involved the modification of a laser ablation electrospray ionization (LAESI) source with a heated gas jet to enable the simultaneous study of polar and nonpolar compounds. The heat-assisted LAESI (HA-LAESI) was tested for analytes of different polarities and sizes, and it was found to protonate compounds ranging from selected PAHs to large proteins with more or less equal efficiency. As compared with LAAPPI and LAESI, the HA-LAESI method ionized a wider range of analytes. Biological tissues were successfully fingerprinted, confirming that neutral and nonpolar analytes can be detected in biological matrices, and HA-LAESI was used in MSI. In conclusion, the work presented in this thesis advances the study of neutral and nonpolar compounds through developments in direct open air surface sampling/ionization mass spectrometry. Like other direct open air surface sampling/ionization mass spectrometry methods, however, the described methods are still in their infantry. Additional work is required to establish them as reliable competitors of more traditional techniques.Massaspektrometria (MS) on herkkä, luotettava ja nopea kemiallisen analytiikan menetelmä, jota käytetään tavallisesti erotustekniikoihin kuten neste- tai kaasukromatografiaan yhdistettynä (LC-MS ja GC-MS). Ennen LC- ja GC-MS analyysejä näytteet yleensä esikäsitellään hitailla ja monivaiheisilla menetelmillä ja analyysin kokonaiskesto on kymmenistä minuuteista jopa muutamaan vuorokauteen. Uusilla suorilla MS:n ionisaatiomenetelmillä näytteitä voidaan tutkia nopeasti ilman esikäsittelyä ja erotusta parhaimmillaan muutamissa sekunneissa. Niillä voidaan myös kerätä tietoa näytteen eri kohdista eli kuvantaa näytettä. Suurin osa suorista MS-ionisaatiomenetelmistä sopii hyvin poolisten yhdisteiden, kuten pienten emäksisten lääkeaineiden tutkimiseen, mutta niillä ei voida havaita yhtä herkästi neutraaleja poolittomia yhdisteitä, kuten steroideja tai tri- ja diglyseridejä. Tämän väitöskirjatyön tarkoituksena olikin kehittää ja käyttää suoria MS-ionisaatiomenetelmiä neutraalien poolittomien yhdisteiden tutkimuksessa. Työssä havaittiin, että vaihtovirtalampulla (rf) voidaan saada parempi ionisaatiotehokkuus mikroilmanpainefotoionisaatiossa (μAPPI) ja desorptioilmanpainefotoionisaatiossa (DAPPI) kuin tasavirtalampulla (dc), joten rf-lamppua käyttämällä saadaan analyyseistä herkempiä. Koska suorissa MS-ionisaatiotekniikoissa havaitaan usein näytteen koostumuksesta johtuvia häiriöitä, työssä kehitettiin uuttomenetelmä parantamaan DAPPI-MS tutkimuksia ja sitä käytettiinkin onnistuneesti mm. lääkeaineiden analysoimiseen jätevedestä ja virtsasta. Tutkimuksessa myös kehitettiin uusi suora, laser-ablaatioon ja fotoionisaatioon perustuva MS-ionisaatiomenetelmä (LAAPPI) neutraalien ja poolittomien yhdisteiden aiempaa tarkempaan kuvantamiseen. Tutkimuksessa näytettiin, että LAAPPI sopii kasvien ja eläinkudosten tutkimiseen, ja sitä käytettiin salvian lehden antioksidanttien ja terpeenien kuvantamiseen. Tutkimuksen viimeisessä osassa muokattiin laser-ablaation sähkösumutusionisaatio (laser ablation electrospray ionization, LAESI) nimistä ionisaatiotekniikkaa kuuman kaasuvirtauksen avulla. Näin pystyttiin tutkimaan samanaikaisesti sekä poolisia, että poolittomia yhdisteitä, mikä ei ole mahdollista aiemmin kehitetyillä tekniikoilla, ja lämpöavusteinen LAESI ionisoikin laajemman yhdisteryhmän kuin vertailumenetelmät. Tässä työssä siis edistettiin neutraalien poolittomien yhdisteiden tutkimusta kehittämällä niille sopivia suoria MS-ionisaatiomenetelmiä. Kuten muut suorat MS-ionisaatiomenetelmät, myös tässä työssä tutkitut tekniikat ovat vasta alkutaipaleellaan ja vaativat lisäparannuksia ja lisätutkimusta, jotta niitä voidaan käyttää rutiininomaisesti LC- ja GC-MS tekniikoiden korvaajina

Mitä sinulle kuuluu? Taustatieto kansio : Työkalu lastensuojelussa

Ajatus opinnäytetyöhön sain työelämästä ja omista työelämän kiinnostuksen kohteista. Toiminnallinen opinnäytetyöni käsittelee nuoren itsensä kuulemiseksi tulemista sekä omaohjaaja-suhdetta. Opinnäytetyön produktin taustalla on työelämälähtöinen tarve kehittää työväline tukemaan sijaishuollossa tehtävää lastensuojelutyötä. Produkti on suunnattu käytettäväksi kaikille Sos – lapsikylän nuorisokodin työntekijöille. Tämä opinnäytetyö toteutettiin vuosina 2013-2014. Opinnäytetyön tarkoituksena oli kehittää työväline tukemaan nuoren taustojen selvittämiseen ja nuoren kuulemiseen. Nuorelle annetaan kansion kautta mahdollisuus tuoda oma mielipiteensä hänen tilanteeseensa ja kansion avulla voidaan luoda tavoitteita nuoren tulevaisuuteen yhdessä omaohjaajan kanssa. Valmiissa produktissa käsitellään nuoren elämää kysymysten ja tehtävien kautta. Raportissa käydään läpi nuoren elämän osa – alueita niiden taustalla olevaa teoriapohjaa. Raportissa käsitellään lastensuojelua, sijaishuoltoa ja produktin valmistamisen vaiheita.The idea of thesis is based on my work and my intrests in work. Thesis handles development of hear-ing youth in their own terms and relationship on their own director. Product of thesis is based on work life and the need to develop tools for support of child welfare. Product is aimed for all the workers in Sos - Lapsikylä. This thesis was produced during years 2013-2014. The main purpose of our thesis was to develope a tool for resolve youth background and hear youth on their own terms. Product gives youth an oppor-tunity to have own opinion on theirs situation and with the product creates opportunity to plan theirs future with help of theirs own director Final product contains questions and tasks of life of a youth. Report explains the sections of product and the basic theory of these sections. Report also includes child welfare, foster care and phases of creating the product

Capillary photoionization: interface for low flow rate liquid chromatography-mass spectrometry

This is the first report on capillary photoionization (CPI) interfacing a liquid chromatograph (LC) and mass spectrometer (MS). A new heated CPI ion source was developed, including a heated transfer capillary, a wide oval-shaped and low-depth ionization chamber with a vacuum ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to increase the photoionization efficiency and thus the sensitivity. As both analytes and eluent are first vaporized and then photoionized inside the CPI ion source between the atmosphere and the vacuum of MS, the ion transfer efficiency into the MS and thus the sensitivity is improved. The effect of the most important operation parameters, the eluent flow rate and temperature of the CPI source, on the signal intensity was studied with selected steroids. The feasibility of LC-CPI-MS/MS for the quantitative analysis of steroids was also studied in terms of linearity, repeatability, and limits of detection. The method showed good quantitative performance and sensitivity down to the low femto-mole level.Peer reviewe

A unique Kelch domain phosphatase in Plasmodium regulates ookinete morphology, motility and invasion

Signalling through post-translational modification (PTM) of proteins is a process central to cell homeostasis, development and responses to external stimuli. The best characterised PTM is protein phosphorylation which is reversibly catalysed at specific residues through the action of protein kinases (addition) and phosphatases (removal). Here, we report characterisation of an orphan protein phosphatase that possesses a domain architecture previously only described in Plantae. Through gene disruption and the production of active site mutants, the enzymatically active Protein Phosphatase containing Kelch-Like domains (PPKL, PBANKA_132950) is shown to play an essential role in the development of an infectious ookinete. PPKL is produced in schizonts and female gametocytes, is maternally inherited where its absence leads to the development of a malformed, immotile, non-infectious ookinete with an extended apical protrusion. The distribution of PPKL includes focussed localization at the ookinete apical tip implying a link between its activity and the correct deployment of the apical complex and microtubule cytoskeleton. Unlike wild type parasites, ppkl(-) ookinetes do not have a pronounced apical distribution of their micronemes yet secretion of microneme cargo is unaffected in the mutant implying that release of microneme cargo is either highly efficient at the malformed apical prominence or secretion may also occur from other points of the parasite, possibly the pellicular pores

Analysis of steroids in urine by gas chromatography-capillary photoionization-tandem mass spectrometry

A new heated capillary photoionization (CPI) ion source design was developed to photoionize analytes inside a transfer capillary between a gas chromatograph (GC) and a mass spectrometer (MS). The CPI setup included a wide, oval-shaped vacuum-ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to maximize photoionization efficiency and thus sensitivity. The source contained a nitrogen housing around the ionization chamber inlet to avoid undesirable hydrolysis and oxidation reactions with ambient air and to maximize the proportion of formed molecular radical cations of analytes. The feasibility of the ion source was studied by analyzing 18 endogenous steroids in urine as their trimethylsilyl (TMS) derivatives with gas chromatography-tandem mass spectrometry (GC-MS/MS). The method was validated and applied to human urine samples. To our best knowledge, this is the first time that a capillary photoionization ion source has been applied for quantitative analysis of biological samples. The GC-CPI-MS/MS method showed good chromatographic resolution (peak half-widths between 3.1 to 5.3 s), acceptable linearity (coefficient of determination between 0.981 to 0.996), and repeatability (relative standard deviation (RSD%) between 5 to 18%). Limits of detection (LOD) were between 2 to 100 pg mL(-1) and limits of quantitation (LOQ) were between 0.05 to 2 ng mL(-1). In total, 15 steroids were quantified either as a free steroid or glucuronide conjugate from the urine of volunteers. The new CPI source design showed excellent sensitivity for analysis of steroids in complex biological samples. (C) 2019 Elsevier B.V. All rights reserved.Peer reviewe

Solid Sampling with a Diode Laser for Portable Ambient Mass Spectrometry

A hand-held diode laser is implemented for solid sampling in portable ambient mass spectrometry (MS). Specifically, a pseudocontinuous wave battery powered surgical laser diode is employed for portable laser diode thermal desorption (LDTD) at 940 nm and compared with nanosecond pulsed laser ablation at 2940 nm. Postionization is achieved in both cases using atmospheric pressure photoionization (APPI). The laser ablation atmospheric pressure photoionization (LAAPPI) and LDTD-APPI mass spectra of sage leaves (Salvia officinalis) using a field-deployable quadrupole ion trap MS display many similar ion peaks, as do the mass spectra of membrane grown biofilms of Pseudomonas aeruginosa: These results indicate that LDTD-APPI method should be useful for in-field sampling of plant and microbial communities, for example, by portable ambient MS. The feasibility of many portable MS applications is facilitated by the availability of relatively low cost, portable, battery-powered diode lasers. LDTD could also be coupled with plasma- or electrospray-based ionization for the analysis of a variety of solid-samples.Peer reviewe

Charge Exchange Reaction in Dopant-Assisted Atmospheric Pressure Chemical Ionization and Atmospheric Pressure Photoionization

The efficiencies of charge exchange reaction in dopant-assisted atmospheric pressure chemical ionization (DA-APCI) and dopant-assisted atmospheric pressure photoionization (DA-APPI) mass spectrometry (MS) were compared by flow injection analysis. Fourteen individual compounds and a commercial mixture of 16 polycyclic aromatic hydrocarbons were chosen as model analytes to cover a wide range of polarities, gas-phase ionization energies, and proton affinities. Chlorobenzene was used as the dopant, and methanol/water (80/20) as the solvent. In both techniques, analytes formed the same ions (radical cations, protonated molecules, and/or fragments). However, in DA-APCI, the relative efficiency of charge exchange versus proton transfer was lower than in DA-APPI. This is suggested to be because in DA-APCI both dopant and solvent clusters can be ionized, and the formed reagent ions can react with the analytes via competing charge exchange and proton transfer reactions. In DA-APPI, on the other hand, the main reagents are dopant-derived radical cations, which favor ionization of analytes via charge exchange. The efficiency of charge exchange in both DA-APPI and DA-APCI was shown to depend heavily on the solvent flow rate, with best efficiency seen at lowest flow rates studied (0.05 and 0.1 mL/min). Both DA-APCI and DA-APPI showed the radical cation of chlorobenzene at 0.05-0.1 mL/min flow rate, but at increasing flow rate, the abundance of chlorobenzene M+. decreased and reagent ion populations deriving from different gas-phase chemistry were recorded. The formation of these reagent ions explains the decreasing ionization efficiency and the differences in charge exchange between the techniques.Peer reviewe

Comparison and imputation-aided integration of five commercial platforms for targeted DNA methylome analysis

Targeted bisulfite sequencing (TBS) has become the method of choice for the cost-effective, targeted analysis of the human methylome at base-pair resolution. In this study, we benchmarked five commercially available TBS platforms-three hybridization capture-based (Agilent, Roche and Illumina) and two reduced-representation-based (Diagenode and NuGen)-across 11 samples. Two samples were also compared with whole-genome DNA methylation sequencing with the Illumina and Oxford Nanopore platforms. We assessed workflow complexity, on/off-target performance, coverage, accuracy and reproducibility. Although all platforms produced robust and reproducible data, major differences in the number and identity of the CpG sites covered make it difficult to compare datasets generated on different platforms. To overcome this limitation, we applied imputation and show that it improves interoperability from an average of 10.35% (0.8 million) to 97% (7.6 million) common CpG sites. Our study provides guidance on which TBS platform to use for different methylome features and offers an imputation-based harmonization solution that allows comparative, integrative analysis

Molecular profile of the rat peri-infarct region four days after stroke: Study with MANF

The peri-infarct region after ischemic stroke is the anatomical location for many of the endogenous recovery processes, and the molecular events in the peri-infarct region remain poorly characterized. In this study, we examine the molecular profile of the peri-infarct region on post-stroke day four, time when reparative processes are ongoing. We used a multiomics approach, involving RNA sequencing, and mass spectrometry-based proteomics and metabolomics to characterize molecular changes in the peri-infarct region. We also took advantage of our previously developed method to express transgenes in the peri-infarct region where self-complementary adeno-associated virus (AAV) vectors were injected into the brain parenchyma on post-stroke day 2. We have previously used this method to show that mesencephalic astrocyte-derived neurotrophic factor (MANF) enhances functional recovery from stroke and recruits phagocytic cells to the peri-infarct region. Here, we first analyzed the effects of stroke to the peri-infarct region on post-stroke day 4 in comparison to sham-operated animals, finding that stroke induced changes in 3345 transcripts, 341 proteins, and 88 metabolites. We found that after stroke genes related to inflammation, proliferation, apoptosis, and regeneration were upregulated, whereas genes encoding neuroactive ligand receptors and calcium-binding proteins were downregulated. In proteomics, we detected upregulation of proteins related to protein synthesis and downregulation of neuronal proteins. Metabolomic studies indicated that in after stroke tissue there is increase in saccharides, sugar phosphates, ceramides and free fatty acids and decrease of adenine, hypoxantine, adenosine and guanosine. We then compared the effects of post-stroke delivery AAV1-MANF delivery to AAV1-eGFP (enhanced green fluorescent protein). MANF administration increased the expression of 77 genes, most of which were related to immune response. In proteomics, MANF administration reduced S100A8 and S100A9 protein levels. In metabolomics, no significant differences between MANF and eGFP treatment were detected, but relative to sham surgery group, most of the changes in lipids were significant in the AAV-eGFP group only. This work describes the molecular profile of the peri-infarct region during recovery from ischemic stroke, and establishes a resource for further stroke studies. These results provide further support for parenchymal MANF as a modulator of phagocytic function.Peer reviewe

Analysis of nitrogen-based explosives with desorption atmospheric pressure photoionization mass spectrometry

RATIONALE: Fast methods that allow the in situ analysis of explosives from a variety of surfaces are needed in crime scene investigations and home-land security. Here, the feasibility of the ambient mass spectrometry technique desorption atmospheric pressure photoionization (DAPPI) in the analysis of the most common nitrogen-based explosives is studied. METHODS: DAPPI and desorption electrospray ionization (DESI) were compared in the direct analysis of trinitrotoluene (TNT), trinitrophenol (picric acid), octogen (HMX), cyclonite (RDX), pentaerythritol tetranitrate (PETN), and nitroglycerin (NG). The effect of different additives in DAPPI dopant and in DESI spray solvent on the ionization efficiency was tested, as well as the suitability of DAPPI to detect explosives from a variety of surfaces. RESULTS: The analytes showed ions only in negative ion mode. With negative DAPPI, TNT and picric acid formed deprotonated molecules with all dopant systems, while RDX, HMX, PETN and NG were ionized by adduct formation. The formation of adducts was enhanced by addition of chloroform, formic acid, acetic acid or nitric acid to the DAPPI dopant. DAPPI was more sensitive than DESI for TNT, while DESI was more sensitive for HMX and picric acid. CONCLUSIONS: DAPPI could become an important method for the direct analysis of nitroaromatics from a variety of surfaces. For compounds that are thermally labile, or that have very low vapor pressure, however, DESI is better suited. Copyright (C) 2016 John Wiley & Sons, Ltd.Peer reviewe