|Title:||Massenspektrometrische Charakterisierung von Phosphorylierungsstellen in mitochondrialen Membranen und Membran/Protein-Komplexen|
|Abstract:||This thesis represents a qualitative and quantitative phosphorylation analysis on mitochondrial proteins of the OM (engl. Outer Membrane) and of membrane/proteincomplexes. After enrichment by IEF, SCX, ERLIC and TiO2 as well as combinations of these methods all together 126 phosphopeptides were detected in mitochondria of Saccharomyces cerevisiae. Beside numerous peptides of OM-proteins 25 phosphosites of the TOM complex, 16 previously undetected, were found amongst. By optimization of loading and elution conditions of tianiumdioxid enrichment phosphosites could also be identified in mitochondria of different mouse organs and in human mitochondria. Out of 66 phosphopeptides characterized in mouse mitochondria solely the three phosphopeptides K.IEDVGpSDEEDDSGKDK.K (Hsp90ab1), R.YGMGTpSVER.A (Pdha1) and M.*AAAVAAAGAGEPLpSPEELLPK.A (Tom22, * = N-terminal acetylation) were found in all organs. The majority of phosphopeptides was only detected in one of the four organs - thereby 25 phosphosites were provided by kidney, followed by heart with 11 and brain/liver with 7 phosphorylated peptides each. In human mitochondria 21 phosphosites were identified, among them phosphopeptides of Tom22 and Tom70. Comparison of the TOM proteins Tom22 and Tom70 through yeast, mouse and human partly showed homologous phosphorylation patterns, leading to potential similar regulation by (de-) phosphorylation. By various enrichment strategies linked to quantification by SILAC differences in the phosphoproteome of yeast strains of CK2 wildtyp and temperature sensitive CK2 mutant could be demonstrated. Alternatively, two labelfree procedures for differential phosphopeptid analysis were presented and proven for their practicability by yeast mitochondria. Especially using synthetic (phospho) peptides and scheduled SRMs turned out to be a very sensitive and reproducible quantitaion method.|
|Appears in Collections:||ISAS|
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