Shotgun proteomics is a widely known technique for identification of protein and peptide sequences from an "in vitro" sample. A tandem mass spectrometer generates tens of thousands of mass spectra which must be annotated with peptide sequences. For this purpose, the similarity search in a database of theoretical spectra generated from a database of known protein sequences can be utilized. Since the sizes of databases grow rapidly in recent years, there is a demand for utilization of various database indexing techniques. We investigate the capabilities of (non)metric access methods as the database indexing techniques for fast and approximate similarity retrieval in mass spectra databases. We show that the method for peptide sequences identification is more than 100x faster than a sequential scan over the entire database while more than 90% of spectra are correctly annotated with peptide sequences. Since the method is currently suitable for small mixtures of proteins, we also utilize a precursor mass filter as the database indexing technique for complex mixtures of proteins. The precursor mass filter followed by ranking of spectra by a modification of the parametrized Hausdorff distance outperforms state-of-the-art tools in the number of identified peptide sequences and the speed of search. The...

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease that causes a disorder of steroidogenesis in the adrenal cortex. This disease is a part of a panel of diseases searched in preclinical nationwide neonatal screening. The methodology is based on measuring the concentration of 17-hydroxyprogesterone (17-OHP) in a dried blood spot using fluorescence immunoassay (FIA). However, this determination is not entirely specific and generates a high rate of false positive results (up to 4.3 %). In this diploma thesis the LC-MS / MS method was developed. This method measures selected steroid hormones involved in cortisol metabolism with respect to the diagnosis of CAH disease. The method was validated and applied to clinical samples, it identified CAH patients from negative controls and significantly reduced the false positivity of neonatal screening results. Compared to the FIA results, the LC-MS / MS method reduced false positivity up to 50 % by evaluating the concentration of 17-OHP. Moreover, by extending the diagnostic algorithm with other measured markers, the reduction was enhanced up to 98%. The developed method is also applicable for the measurement of serum and plasma samples, respectively, and has become a part of the confirmation tests for suspected CAH screening findings. Key...

The goal of my diploma thesis was finding a suitable method for unifying score values from various protein identification search tools in MS/MS mass spectrometry into one single score value. Data coming from the output of mass spectrometer are processed in two independent search tools Mascot and X!Tandem. These were selected especially for their wide usage in proteomic labs. Both results are evaluated through newly designed function and unified by single valued score clearly identifying found proteins. Newly designed scoring value is called Matascore and function producing this score was implemented in MATLAB. Function and its results were successfully tested by real data available in public databases on the Internet.

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Ultraviolet radiation (UVR) in solar light is important for skin biology. It is involved in the development acute and chronic skin inflammation, aging and cancer, causing erythema, tanning and local or systemic immunosuppression. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are considered anti- inflammatory and could reduce the damage caused by overexposure to UVR. Although, n-3 PUFA have been considered as photoprotective agents, their exact mechanisms of action is not completely understood. The aim of the work is to determine the effect of UVR and the n-3 PUFA eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on human skin cells (in vitro study), specifically on: cell viability, apoptosis and their metabolism through the cyclooxygenase and lipoxygenase pathways. Also, to study the cellular incorporation and effect of n-3 PUFA on the fatty acid profile of skin cells. A clinical study was undertaken to assess the incorporation of n-3 PUFA supplements in human skin. A clinical study was performed in 40 healthy women (active group) supplemented with 4g/day of EPA (70%) and DHA (10%) and 40 healthy women (placebo group) supplemented with 4g/day of glyceryl tricoprylate coprate (GTCC). After 3 months, both blood samples and skin punch biopsies were collected and analysed for fatty acids by gas chromatography (GC). HaCaT keratinocytes and 46BR.1N fibroblasts were cultured and treated with 10 and 50μM of either EPA, or DHA or oleic acid (OA) for 72h and exposed to 15 and 50 mJ/cm2. Cell viability was measured by the MTT assay and cell apoptosis by a colorimetric method, at 24h post UVR. Cells and culture media were analysed by GC and liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) to assess cellular fatty acids and production of eicosanoids. The clinical a study showed that in RBC saturated fatty acids (SFA) (44.27±7.43%) were the main fatty acid group followed by n-6 PUFA (29.61±5.53%). While in dermal tissue monounsaturated fatty acids (MUFA) (58.90±9.80%) was the main fatty acid group followed by SFA (27.06±6.78%). A significant increase in EPA, DHA and docosapentaenoic acid (DPA) was observed in RBC but only EPA was significantly increased in the dermis post n-3 PUFA supplementation. . The viability of HaCaT keratinocytes and 46BR.1N fibroblasts decreased post UVR and this was further reduced post PUFA treatment. Cell apoptosis increased when cells were exposed to UVR and further increased when cells were treated with EPA and DHA. . In HaCaT keratinocytes MUFA (54.22±8.82%) was the main fatty acid group followed by FAS (37.11±.9.16%), while SFA (51.94±8.68%) was the main group followed by MUFA (27.07±4.79) in 46BR.1N. Treated both cells with EPA and DHA showed significant increased in cellular EPA, DPA and DHA. 46BR.1N fibroblasts produced higher levels of prostaglandins (PG) compared to HaCaT keratinocytes: PGE2 and PGD2 were the main PG in both HaCaT (7.96±3.18 and 1.48±1.19 pg/million cell; respectively) and 46BR.1N with (44.2±23.00 and 17.1±9.71 pg/million cell; respectively). Significant increase in PGE1 and PGE2 occurred when cells were exposed to 15mJ/cm2 UVR. Treatment with n-3 PUFA decreased the level of PGE1 and PGE2, and increase production PGE3 at the baseline and post UVR. Both cell lines produced hydroxy fatty acids and the concentration of these mediators was higher in 46BR.1N than HaCaT. The concentrations of these mediators were significant increased post UVR: treatment with n-3 PUFA decreased the level of HODE and HETE, and increase production of HEPE and HDHA at baseline and post UVR. Overall, n-3PUFA treatment led to increases in the content of EPA and DHA on RBC, dermal tissue and human skin cell lines. EPA and DHA in skin cell lines appear to offer protection by increasing cellular apoptosis, decreasing inflammatory mediators specifically PGE2 and 12-HETE, and increasing anti-inflammatory mediators such as PGE3, 15-HEPE and 17-HDHA.

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<p>During the past fifty years, mass spectrometry, often hyphenated with chromatography, has developed into the most widely used technique for the quantitative and qualitative analysis of increasingly complex mixtures of (bio)organic molecules.</p> <p>One important aspect of this development concerns the relationship between the structure (atom connectivity) of a molecule and the mass spectrum obtained by electron ionization (EI). In this context, from 1960 - 1990, a wealth of studies has appeared that uses a variety of novel experimental techniques, often in conjunction with isotope labelling, to probe the structure, stability, reactivity and dissociation characteristics of the radical cations generated by EI of various classes of molecules. One highlight was the discovery of surprisingly stable distonic ions and the role they play in the dissociation chemistry of ionized molecules.</p> <p>However, mechanistic proposals based upon experimental observations can often only be considered as tentative. Synergy between experiment and theory would be ideal to remedy this situation, but it was not until recent spectacular advances in computer technology and software that this approach could be implemented. It has led to the growing realization that many rearrangement reactions of radical cations in the rarefied gas-phase involve catalysis. Proton-transport catalysis (PTC) is a prime example : here, a neutral species induces an ion to isomerize via hydrogen-bridged radical cations (HBRCs) as intermediates. An exemplary case described in this thesis concerns the ion-molecule reaction of the cyanamide ion with a single H₂O molecule : experiment and theory indicate that the H₂O molecule catalyzes the swift transformation of NH₂-CN^·⁺ into the more stable carbodiimide ion HN=C=NH^·⁺.</p> <p>The thesis exploits the synergy of tandem mass spectrometry and computational chemistry to study the role of catalysis in the association and dissociation reactions of several systems of radical cations. During these studies, a new type of a catalyzed reaction was discovered: "ion-catalysis", where an organic cation promotes the otherwise prohibitive rearrangement of a neutral. Ion-catalysis is proposed to explain the unexpected loss of NH₂O^· from low-energy N-hydroxyacetamide ions CH₃C(=O)NHOH^·⁺ : the molecular ion rearranges into the HBRC [O=C-C(H₂)--H--N(H)OH]^·⁺ whose acetyl (cation) component catalyzes the transformation NHOH^· --> NH₂O^·. Another highlight involves a hybrid reaction, in which both the ion and the neutral component of an incipient HBRC catalyze one another to rearrange into more stable isomers.</p> <p>Catalysis may also play an important role in astrochemistry and a question addressed in this context is whether pyrimidine, a key component of DNA, may be generated by ion-molecule reactions. It appears that the acrylonitrile ion (AN) does not react with HCN to produce ionized pyrimidine, instead it isomerizes by PTC. However, the reaction of the ion with its neutral counterpart does not involve catalysis, but rather cyclization into the pyrimidine ion ! A related topic concerns the structures of covalently bound dimers of the ubiquitous interstellar molecules HCN and HNC. Neutralization-Reionization Mass Spectrometry in conjunction with model chemistry calculations leaves little doubt that the elusive dimers HN=C=C=NH and HC=N-C=NH are kinetically stable in the rarefied gas-phase, whereas HC=N-N=CH is not.</p> <p>The structure of ions may also be probed by interactions with selected neutral molecules rather than dissociative collision experiments (MS/MS). An exciting case involves the differentiation of isomeric heterocyclic ions by ion-molecule reactions with dioxygen. Here, too, model chemistry calculations play an essential role in understanding the mechanism and the scope of the reaction.</p> / Doctor of Philosophy (PhD)