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.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:24415 |
| Date | 22 November 2004 |
| Creators | Shevchenko, Anna |
| Contributors | Rödel, G., Hoflack, B., Jensen, O. N. |
| Publisher | Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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