Die schnelle, sog. Saltatorische Erregungsleitung bei den Vertebraten wird durch eine kompakte, um das Axon gewickelte isolierende Schicht, dem Myelin, ermöglicht. Diese Myelinhülle wird von spezialisierten Gliazellen gebildet. Bei der Myelinisisierung wickeln sich deren abgeflachte Membranfortsätze mehrfach konzentrisch um die Axone und bilden nach Verdichtung die typische kompakte, multi-lammelare Struktur des Myelins. Innerhalb dieser Struktur unterscheidet man zwei unterschiedliche Bereiche: Die aus der extrazellulären Apposition gebildete Intraperiod Line und die aus der cytosolischen Apposition gebildete Major Dense Line . Ausschließlich innerhalb der Major Dense Line des ZNS der Fische findet man ein Protein, das nach seinem Molekulargewicht als 36K bezeichnet wird. Es ist nicht glykolisiert und scheint mit der Myelinmembran assoziiert zu sein. Immunologische Untersuchungen zeigten, dass 36K mit keinem der polyklonalen Antikörper gegen eines der bekannten Myelinproteine reagierte. Ebenso zeigten erste Datenbank-Analysen überraschenderweise keine Homologien zu den Myelinproteinen, sondern zu den NAD(P)(H)-abhängigen Oxidoreduktasen. Im Rahmen der vorliegenden Arbeit wurde versucht über einen Dehydrogenase-Assay sowohl eine entsprechende Aktivität als auch ein mögliches Substrat zu identifizieren. Alternativ wurde für das membranassoziierte 36K, das neben IP1 & 2 und Basischen Myelinprotein (MBP) eines der Hauptmyelinproteine im ZNS der Fische darstellt, eine Funktion als Strukturprotein innerhalb der Major Dense Line vermutet. Aufgrund der Sequenzähnlichkeiten von 36K zu den NAD(P)(H)-abhängigen Oxidoreduktasen wurde dabei auch die Beeinflussbarkeit durch Nukleotide geprüft. Des Weiteren wurde im Rahmen dieser Dissertation erstmals versucht, Protein-Komplexe aus dem ZNS-Myelin nativ aufzutrennen und deren Komponenten zu identifizieren.

The major goal of this study was to develop an experimental proteomics approach for deciphering protein complexes and protein interaction networks in the budding and fission yeasts. Key steps of the employed analytical routine, including the purification of complexes and mass spectrometric identification of their subunits, were investigated in detail. Archiving, storage and handling of polyacylamide gels, visualization of protein bands and their effect on the efficiency of in-gel digestion and mass spectrometric identification of proteins were quantitatively evaluated. It was further demonstrated that a combination of several mass spectrometric techniques based on MALDI and ES ionization provided complementary data and enabled comprehensive characterization of protein digests. The optimized analytical procedures were employed in deciphering protein complexes and protein interaction networks in the budding and fission yeasts. A combination of Tandem Affinity Purification (TAP) and mass spectrometric identification of gel separated protein subunits is generic and robust strategy that provided accurate and reproducible data. The evaluation of TAP success rate, reproducibility and typical protein background presented in this work is based on TAP tagging and immunoprecepitation of 75 genes in S. cerevisiae and 22 in S. pombe. The molecular composition of characterized protein complexes was compared with protein-protein interactions uncovered by other established methods, such as yeast two hybrid screens or proteome-wide purification of protein complexes. We found that repetitive purification of protein complexes using different subunits as baits is crucially important for confident charting of proteomic environments. Accurate dissection of individual protein complexes and identification of their proteomic hyperlinks enabled to consider proteomic environments in the phylogenetic perspective and paved the way to reliable projection of proteomics data obtained in lower eukaryotic model organisms to higher eukaryotes, including humans.

The major goal of this study was to develop an experimental proteomics approach for deciphering protein complexes and protein interaction networks in the budding and fission yeasts. Key steps of the employed analytical routine, including the purification of complexes and mass spectrometric identification of their subunits, were investigated in detail. Archiving, storage and handling of polyacylamide gels, visualization of protein bands and their effect on the efficiency of in-gel digestion and mass spectrometric identification of proteins were quantitatively evaluated. It was further demonstrated that a combination of several mass spectrometric techniques based on MALDI and ES ionization provided complementary data and enabled comprehensive characterization of protein digests. The optimized analytical procedures were employed in deciphering protein complexes and protein interaction networks in the budding and fission yeasts. A combination of Tandem Affinity Purification (TAP) and mass spectrometric identification of gel separated protein subunits is generic and robust strategy that provided accurate and reproducible data. The evaluation of TAP success rate, reproducibility and typical protein background presented in this work is based on TAP tagging and immunoprecepitation of 75 genes in S. cerevisiae and 22 in S. pombe. The molecular composition of characterized protein complexes was compared with protein-protein interactions uncovered by other established methods, such as yeast two hybrid screens or proteome-wide purification of protein complexes. We found that repetitive purification of protein complexes using different subunits as baits is crucially important for confident charting of proteomic environments. Accurate dissection of individual protein complexes and identification of their proteomic hyperlinks enabled to consider proteomic environments in the phylogenetic perspective and paved the way to reliable projection of proteomics data obtained in lower eukaryotic model organisms to higher eukaryotes, including humans.