| Abstract: | The arachidonic acid (ARA) cascade is a key regulatory pathway where eicosanoids and other oxylipins are formed via the oxidation of polyunsaturated fatty acids. These oxylipins are involved in controlling physiological functions such as vascular tone, blood clotting and immune regulation. Changes in the oxylipin pattern can be caused by direct enzyme inhibition or modulated gene expression. In order to understand the effect mechanisms modulating the ARA cascade it is essential to not only investigate the metabolite, i.e. oxylipin, concentrations but also the abundances of the enzymes involved in their formation. For this reason, a quantitative targeted proteomics method for the ARA cascade was developed within this thesis. A detailed workflow for method development was established where enzyme/protein levels are measured via sequence-specific, representative peptides chosen on the basis of several criteria. With liquid chromatography-tandem mass spectrometry-based analysis, the cyclooxygenase (COX) pathway of the ARA cascade was investigated in human colon carcinoma cell lines and primary macrophages where strong correlations were found between the oxylipins formed via the COX pathway and the COX-2 abundance. The modulating effects of polyphenols on the 5-lipoxygenase (LOX) pathway of the ARA cascade were investigated in cell-free enzyme assays and human neutrophils. Resveratrol, its dimer ?-viniferin and a resveratrol imine analogue directly inhibited the 5-LOX with micromolar potencies and modulated the total oxylipin pattern in the neutrophils with remarkably different effects. Genistein did not act as a 5-LOX inhibitor in the cell-free assay but the modulated oxylipin pattern in the cells suggests a global cellular interference. The enzymes of the LOX pathway were included in the targeted proteomics method which together with an extended targeted oxylipin metabolomics platform enables a sensitive multi- omics analysis of the ARA cascade in a single sample. The comparison of the multiple reaction monitoring (MRM) detection and the MRM^-based approach with an additional fragmentation step demonstrated that MRM is more favorable in targeted proteomics due to its higher sensitivity, greater linear range and higher multiplexing capacities. The analysis with the multi- omics approach showed that the 5 LOX protein is induced together with its product formation during the differentiation of human monocytic THP 1 cells to macrophages. M1-like human primary macrophages were also characterized by the abundance of 5-LOX and its activating protein (FLAP), while 15-LOX and 15-LOX-2 dominated the protein pattern of the M2 like macrophages accompanied by high levels of the oxylipins formed via these enzymes. The developed methodology allows mechanistic investigation of a modulation of the ARA cascade as demonstrated by lipo-poly-saccharide stimulation as well as pharmaceutical treatment of the cells. Overall, with this thesis, a new quantitative proteomics strategy for the enzymes of the ARA cascade is established which can be easily extended to further proteins. The use of this technique in combination with targeted oxylipin metabolomics has provided new insights into the mechanisms involved in the modulation of the ARA cascade in human immune cells. |
