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BioZ, TUDresden

The core proteome of S. cerevisiae has been mapped using knock-in tag methodology EMBL/CellZome, Heidelberg Krogan…Greenblatt, Toronto. BioZ, TUDresden. Seeking a rational organization of eukaryotic proteomes:.  document the Core Proteome:

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BioZ, TUDresden

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  1. The core proteome of S. cerevisiae has been mapped using knock-in tag methodology EMBL/CellZome, Heidelberg Krogan…Greenblatt, Toronto BioZ, TUDresden

  2. Seeking a rational organization of eukaryotic proteomes: document the Core Proteome: the constellation of biochemically stable complexes and free proteins • onto this scaffold the mapping of weaker (real) transient, regulated and cell-type specific interactions can be organized (2-Hy information?) accurate documentation of protein-protein interactions is essential!

  3. Navigating the core proteome by sequential tagging: Pijnappel,W.W.M., Schaft,D., Roguev, A., Shevchenko, A., Wilm, M., Rigaut, G., Séraphin, B., Aasland, R., and Stewart, A.F. The S. cerevisiae Set3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program. Genes and Development 15, 2991-3004. (2001) Roguev, A., Schaft, D., Shevchenko, A., Pijnappel, W.W.M., Wilm, M., Aasland, R. and Stewart, A.F. The S. cerevisiae Set1 complex includes an Ash2-like protein and methylates histone 3 lysine 4 EMBO J. 20, 7137-7148, (2001).

  4. LC MS/MS Dionex Ultimate nanoLC + ThermoElectron LTQ ion trap Anna and Andrej Shevchenko Matthias Wilm Matthias Mann Bertrand Seraphin.

  5. the core proteome is mappable! accuracy depended upon phsiological expression levels (knock-ins) ES cells are the best venue for mapping the core mammalian proteome - homologous recombination - biochemistry - access to cell types via differentiation in vitro or the mouse The core proteome of S. cerevisiae has been mapped using knock-in tag methodology EMBL/CellZome, Heidelberg Krogan…Greenblatt, Toronto - maximize informational base to improve mouse experimentation - another use of ES cells to reduce animal experimentation Start with the transcriptome - combined use of tag for generic ChIP on chip BioZ, TUDresden

  6. A generic multi-purpose allele design for tagging/functional analyses

  7. Genetrap style targeting -- add FlEx

  8. Problems of the (CaTZZ) TAP-Tag The ZZ domain of protein A bind to endogenous IgG Elution of the Calmodulin Binding domain from the beads TEV is not the optimal protease Knock-in of a protein tag in ES cells & mice Testa et al, (2003) Nature Biotechnology, 21, 443-447 Giuseppe Testa Misho Sarov

  9. Development of new Tag combinations New double cassettes

  10. “In vitro” pull down 20 ng/l GST-TAP mouse brain extract 5mg/ml 1:250 dilution (abundant protein) Coomassie blue stained gel

  11. Development of multi-purpose alleles Further development of new tags Protocol development - large scale growth of ES cells - large scale extracts from mouse tissues - affinity IPs from mammalian extracts Evaluation of new tags in ES cells Mapping the mammalian core proteome - progress

  12. 3. Improve sensitivity of MS identifications to reduce scales/costs of mammalian purifications. Higher yield of protein digestion - gel-free shotgun LC MS/MS analysis Improved peptide detection - use of fast, high capacity 2D ion trap mass spectrometer • Improved database searches • use of sequence similarity searching tools for mining genomic and EST • databases Mapping the mammalian core proteome Towards an optimized strategy

  13. Acknowledgements Shevchenko group: MPI-CBG, Dresden Vineeth Surendranath* Patrice Waridel * Youri Kravatsky* *supported by BMBF Henrik Thomas Shamil Sunyaev (Harvard Medical School, Boston) David Drechsel (MPI-CBG, Dresden) A. F. Stewart group: Technical University of Dresden Genomics, BioZ Senming Zhao* Michael Sarov * Daniel Schaft Assen Roguev Pim Pijnappel Sandra Lubitz Stefan Glaser *supported by BMBF Frieder Schwenk (Artemis Pharmaceuticals, Koeln)

  14. Open issues recombineering strategy the ideal protein tag

  15. 2. Obtain the BAC from the genome resources 3. Subclone according to design 4. Insert multi-purpose/conditional cassettes Rapid generation of targeting constructs v2005 1. Design targeting construct from genome sequence & wrt expression in ES cells (~70%), expressed = promoter trap (‘targeted trapping’) not expressed = large with predesigned Southern strategy EUCOMM dedicated software for targeting design

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