Efficient Calculation of Accurate Masses of Isotopic Peaks

This paper presents a new method for calculating accurate masses of isotopic peaks. It is based on breaking the calculation into a binary series of calculations. The molecule is built up by a series of such calculations. At each step the accurate masses are calculated as a probability weighted sum of the masses of the contributing peaks. The method is computationally efficient and accurate for both mass and relative abundance

Methods and tools for mass spectrometric lipidome analysis

The analysis of lipid compositions from biological samples has become increasingly important. Lipids have a role in cardiovascular disease, metabolic syndrome and diabetes. They also participate in cellular processes such as signalling, inflammatory response, aging and apoptosis. Also, the mechanisms of regulation of cell membrane lipid compositions are poorly understood, partially because a lack of good analytical methods. Mass spectrometry has opened up new possibilities for lipid analysis due to its high resolving power, sensitivity and the possibility to do structural identification by fragment analysis. The introduction of Electrospray ionization (ESI) and the advances in instrumentation revolutionized the analysis of lipid compositions. ESI is a soft ionization method, i.e. it avoids unwanted fragmentation the lipids. Mass spectrometric analysis of lipid compositions is complicated by incomplete separation of the signals, the differences in the instrument response of different lipids and the large amount of data generated by the measurements. These factors necessitate the use of computer software for the analysis of the data. The topic of the thesis is the development of methods for mass spectrometric analysis of lipids. The work includes both computational and experimental aspects of lipid analysis. The first article explores the practical aspects of quantitative mass spectrometric analysis of complex lipid samples and describes how the properties of phospholipids and their concentration affect the response of the mass spectrometer. The second article describes a new algorithm for computing the theoretical mass spectrometric peak distribution, given the elemental isotope composition and the molecular formula of a compound. The third article introduces programs aimed specifically for the analysis of complex lipid samples and discusses different computational methods for separating the overlapping mass spectrometric peaks of closely related lipids. The fourth article applies the methods developed by simultaneously measuring the progress curve of enzymatic hydrolysis for a large number of phospholipids, which are used to determine the substrate specificity of various A-type phospholipases. The data provides evidence that the substrate efflux from bilayer is the key determining factor for the rate of hydrolysis.Näytteiden lipidiseosten koostumuksen mittaaminen on tullut yhä tärkeämmäksi. Lipideillä on rooli sydän- ja verisuonitaudeissa, metabolisessa oireyhtymässä ja diabeteksessä. Lipidit myös osallistuvat moniin solun prosesseihin kuten signaalinvälitykseen, tulehdusreaktioon, vanhenemiseen ja apoptoosiin. Hyviä mittausmenetelmiä tarvitaan myös solun kalvojen lipidikoostumuksen säätelymekanismien tutkimiseen. Massaspektrometria on avannut uusia mahdollisuuksia lipidinäytteiden analysiontiin suuren erottelukykynsä ja herkkyytensä ansiosta. Näytteiden sähkösumutusmenetelmä sekä laitteiden jatkuva kehittyminen ovat mullistaneet lipidiseosten koostumuksen analytiikan. Sähkösumutusmenetelmän etuna on se että lipidit eivät hajoa ionisoinnin aikana, jolloin monikomponettisten seosten analysionti helpottuu. Lipidisignaalien epätäydellinen erottuminen, eri lipidien erilaiset vasteet sekä mittauksissa syntyvän datan suuri määrä vaikeuttavat monikomponenttisten seosten massaspektrometrista analysointia. Tämän vuoksi mittausdatan analysointi täytyy automatisoida. Tämän väitöskirjatyön aiheena on monikomponenttisten lipidiseosten massaspektrometristen analyysimenetelmien kehitys. Työssä on tutkittu sekä kokeellisia että laskennallisia menetelmiä. Ensimmäinen osatyö käsittelee lipidiseosten analysointia massaspektrometrillä ja selvittää kuinka fosfolipidien ominaisuudet ja pitoisuus vaikuttavat niiden vasteeseen massaspektrissä. Toisessa artikkelissa kuvataan uusi laskennallinen menetelmä isotooppipiikkijakaumien laskentaan. Kolmannessa artikkelissa julkaistaan monikomponenttisten lipidiseosten analysointiin kehitetyt ohjelmistot sekä vertaillaan erilaisia laskennallisia menetelmiä samankaltaisten lipidien päällekkäisten signaalien erottamiseen. Neljännessä osatyössä sovelletaan kehitettyjä menetelmiä useiden fosfolipiden seoksen yhtäaikaisen hydrolyysireaktion etenemiskäyrien mittaukseen, joiden avulla määritetään A-tyypin fosfolipaasien substraattispesifisyys. Tulosten perusteella voidaan myös päätellä substraatin irtautumisen kaksoiskalvosta olevan tärkein hydrolyysinopeuteen vaikuttava tekijä

Endophytic Bacillus and Pseudomonas spp. Modulate Apple Shoot Growth, Cellular Redox Balance, and Protein Expression Under in Vitro Conditions

Interactions between host plants and endophytic microorganisms play an important role in plant responses to pathogens and environmental stresses and have potential applications for plant stress management under in vitro conditions. We assessed the effect of endophytic bacteria on the growth and proliferation of domestic apple cv. Gala shoots in vitro. Further, a model apple cell suspension system was used to examine molecular events and protein expression patterns at an early stage of plant–endophyte interaction. Among the seven strains used in the study, Bacillus spp. strains Da_1, Da_4, and Da_5 and the Pseudomonas fluorescens strain Ga_1 promoted shoot growth and auxiliary shoot proliferation. In contrast, Bacillus sp. strain Oa_4, P. fluorescens strain Ga_3 and P. orientalis strain G_12 inhibited shoot development. In the cell suspension, the effects of the association between endophytic bacteria and plant cells were specific to each strain. Modulation of the cellular redox balance was monitored in the apple cells using a 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) probe, and strain-specific effects were observed that correlated with the in vitro shoot development results. Proteomic analysis revealed differences in protein expressions in apple cells co-cultivated with different Bacillus spp. strains that had contrasting effects on cellular redox balance and shoot development. The Bacillus sp. strain Da_4, which enhanced shoot development and oxidation of H2DCFDA, induced differential expression of proteins that are mainly involved in the defense response and regulation of oxidative stress. Meanwhile, treatment with Bacillus sp. strain Oa_4 led to strong upregulation of PLAT1, HSC70-1 and several other proteins involved in protein metabolism and cell development. Taken together, the results suggest that different cell signaling and response events at the early stage of the plant–endophyte interaction may be important for strain-dependent regulation of cellular redox balance and development of shoot phenotype

The Comparison of Constant and Dynamic Red and Blue Light Irradiation Effects on Red and Green Leaf Lettuce

In this study, we sought to evaluate and compare the effects of constant and dynamic lighting on red and green leaf lettuce (Lactuca sativa L. Red Cos and Lobjoits Green cos) cultivated in a controlled environment. Plants were illuminated with the combination of red 662 and 638 nm, blue 452 nm, and far-red 737 nm at 16 h photoperiod and constant daily light integral (DLI) of each component. Five constant or dynamic lighting treatments were performed: (BR) constant flux of both B452 and R662; (B*R) constant flux of R662, but the DLI of B452 condensed in 8 h in the middle of photoperiod doubling the PPFD of blue light; (BR*) constant flux of B452, but the DLI of R662 light condensed in the middle of photoperiod; (BdynR) constant flux of R662, but the flux of B452 varies in the sinusoidal profile during 16 h photoperiod, imitating diurnal increase and decrease in lighting intensity; and (BRdyn) constant flux of B452, but the flux of R662 varies in sinusoidal profile. The lettuce’s response to dynamic lighting strategies was cultivar specific. Dynamic lighting strategies, mimicking natural lighting fluctuations, did not have a remarkable effect on photosynthesis and antioxidative parameters, but the dynamic flux of blue light component had a pronounced effect on higher macro and microelement contents in lettuce leaves

Treatment of Arabidopsis thaliana seeds with cold plasma induces changes in seedling and plant development and protein expression

Successful seed germination and seedling establishment are critical steps in agricultural production and in the maintenance of natural ecosystems. Seed treatment cold plasma (CP), is an emerging eco-agricultural technology that has been suggested to stimulate plant growth. In addition to sterilization effect, treatment of seeds with CP has been shown to have effect on a broad spectrum of plant development and physiological processes. Although the body of information on the biological effects of the CP is growing, the complex biological mechanisms involved in the plant response remains vague. To gain an insight into the cellular processes underlying response of Arabidopsis thaliana to the CP seed treatment, seedlings and plant morphological changes, production of reactive oxygen species (ROS) and differential protein expression was assessed. The most prominent morphological effect was observed after the seed treatment for 0,5-4,5 min at 3 mm distance from the dielectric barrier discharge (DBD) plasma source at 60-80% air humidity, after the seeds were stored for 7 days at 25 oC. The treated plants had more compact rosette and longer flower stems. Assessment of ROS production in germinated seedlings using nitro blue tetrazolium and 3,3′- diaminobezidine staining revealed increase in accumulation of superoxide and H2O2. The 2Delectrophoresis analysis revealed 34 and 4 proteoforms differentially expressed after CP treatment in seedlings and leaves, respectively. Proteins involved in biological processes of response to stress and metabolism were identifiedBiochemijos katedraGamtos mokslų fakultetasLietuvos agrarinių ir miškų mokslų centro Sodininkystės ir daržininkystės institutasVytauto Didžiojo universiteta

Constitutive and cold acclimation-regulated protein expression profiles of Scots Pine seedlings reveal potential for adaptive capacity of geographically distant populations

Geographically distant Scots pine (Pinus sylvestris L.) populations are adapted to specific photoperiods and temperature gradients, and markedly vary in the timing of growth patterns and adaptive traits. To understand the variability of adaptive capacity within species, molecular mechanisms that govern the physiological aspects of phenotypic plasticity should be addressed. Protein expression analysis is capable of depicting molecular events closely linked to phenotype formation. Therefore, in this study, we used comparative proteomics analysis to differentiate Scots pine genotypes originating from geographically distant populations in Europe, which show distinct growth and cold adaptation phenotypes. Needles were collected from 3-month-old seedlings originating from populations in Spain, Lithuania and Finland. Under active growth-promoting conditions and upon acclimation treatment, 65 and 53 differentially expressed proteins were identified, respectively. Constitutive protein expression differences detected during active growth were associated with cell metabolism and stress response, and conveyed a population-specific adaptation to the distinct climatic conditions. Acclimation-induced protein expression patterns suggested the presence of a similar cold adaptation mechanism among the populations. Variation of adaptive capacity among the genotypes was potentially represented by a constitutive low level of expression of the Ser/Thr-protein phosphatase, the negative regulator of the adaptive response. Also, overall less pronounced acclimation-induced response in seedlings from the Spanish population was observed. Thus, our study demonstrates that comparative proteomic analysis of young conifer seedlings is capable of providing insights into adaptation processes at the cellular level, which could help to infer variability of adaptive capacity within the plant speciesLietuvos agrarinių ir miškų mokslų centrasMiškų ir ekologijos fakultetasVytauto Didžiojo universiteta

Cold plasma treatment of sunflower seeds modulates plant-associated microbiome and stimulates root and lateral organ growth

Cold atmospheric pressure (CP) plasma irradiation of seeds has been shown to promote plant growth, but the molecular basis of this phenomenon is poorly understood. In our study, optimum irradiation of common sunflower seeds using a dielectric barrier discharge CP device stimulated growth of sunflower lateral organs and roots by 9–14% compared to the control. Metagenomic analysis revealed that the structure of plant-associated bacterial assembly was greatly modified upon CP treatment and could be attributed to the antimicrobial effect of CP-generated reactive species. The treatment resulted in the domination of spore forming Mycobacterium sp. in the above-ground tissues of the seedlings. While the overall bacterial diversity in the roots was barely affected, the CP-induced shift in microbial composition is the likely basis for the observed seedling root growth stimulation and the long-term effect on lateral organ growth and could be mediated by increase in water uptake and/or direct root signaling. Low amplitude protein abundance differences were detected in the roots of the emerging seedlings that are characteristic to low intensity stress stimuli response and could be linked to the changes in plant-associated microbiome upon CP treatmen

Biochip Surfaces Containing Recombinant Cell-Binding Domains of Fibronectin

Surface immobilization and characterization of the functional activity of fibronectin (Fn) type-III domains are reported. The domains FnIII9-10 or FnIII10 containing the RGD loop and PHSRN synergy site were recombinantly produced and covalently bound to chemically activated PEG methacrylate (MA) hydrogel coatings by microcontact printing. Such fabricated biochip surfaces were 6 mm in diameter and consisted of 190 µm wide protein stripes separated by 200 µm spacing. They were analyzed by imaging null ellipsometry, atomic force microscopy and fluorescence microscopy. Also, the coatings were tested in human foreskin fibroblast and HeLa cultures for at least 96 h, thus evaluating their suitability for controlled cell adhesion and proliferation. However, while HeLa cultures were equally well responsive to the FnIII9-10, FnIII10 and Fn surfaces, the fibroblasts displayed lower cell and lower focal adhesion areas, as well as lower proliferation rates on the Fn fragment surfaces as compared to Fn. Nevertheless, full functional activity of the fibroblasts was confirmed by immunostaining of Fn produced by the cells adherent on the biochip surfaces. The observed interaction differences that were either cell type or surface composition-dependent demonstrate the potential use of specifically engineered Fn and other ECM protein-derived domains in biochip architectures

Changes in germination, seedling growth, phytohormone content and proteome induced in Helianthus annuus by pre-sowing seed treatment with stressors

Seed treatment with cold plasma (CP) or electromagnetic field (EMF) is a modern eco-agricultural technology for stimulation of plant germination and performance. Numerous studies demonstrated the effectiveness of such treatments for enhancing germination and growth of a large variety of crops, however the molecular basis of seed response to treatments remains elusive. In order to gain an insight into the molecular mechanisms underlying effect of stressors on plant seeds, we estimated changes induced in differential protein expression of the model plant – sunflower (Helianthus annuus). We studied the effects of pre-sowing seed treatment, using vacuum (7 min), radio-frequency EMF (5-15 min) and CP (2-7 min), on germination and growth of the confectionary cultivar “Nyķrségi fekete”. The germination tests were performed both in vitro and in substrate and the obtained results indicated that the treatments with CP and EMF had no effect on germination yield while vacuum and EMF (10 and 15 min) treatments increased germination rate. We demonstrate that seed treatments with CP induce decrease in seed phytohormone content – decrease in content of abscisic acid and increase in content of gibberelins. Treatments had effect on morphology of seedlings that developed from the CP (7 min) and EMF (15 min) treated seeds resulting in reduced height/weight of stems and increased weight of leaves, respectively. [...]Aplinkotyros katedraBiochemijos katedraGamtos mokslų fakultetasLietuvos agrarinių ir miškų mokslų centrasLietuvos agrarinių ir miškų mokslų centro Sodininkystės ir daržininkystės institutasVytauto Didžiojo universiteta