Upload
constance-mills
View
221
Download
0
Embed Size (px)
DESCRIPTION
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, (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, , (2001).
Citation preview
BioZ,TUDresden
The core proteome of S. cerevisiae has been mapped using knock-in tag methodology
EMBL/CellZome, HeidelbergKrogan…Greenblatt, Toronto
document the Core Proteome: the constellation of biochemically stable complexes and free proteins
Seeking a rational organization of eukaryotic proteomes:
accurate documentation of protein-protein interactions is essential!
onto this scaffold the mapping of weaker (real) transient, regulated and cell-type specific interactions can be organized (2-Hy information?)
TAP-
TAP-
TAP-
1st round 2nd round 3rd round
A B
A
C
TAP-
TAP-
B
C
B
A
C
B
A
Cx
y
xC
x
yTAP- y
Complex I
Complex II
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).
TEV-protease
Calmodulin-binding-peptide
IgG
Protein A
Calmodulin
The TAP method
Bertrand Seraphin.
Anna and Andrej ShevchenkoMatthias WilmMatthias Mann
LC MS/MS
Dionex Ultimate nanoLC + ThermoElectron LTQ ion trap
BioZ,TUDresden
The core proteome of S. cerevisiae has been mapped using knock-in tag methodology
EMBL/CellZome, HeidelbergKrogan…Greenblatt, Torontothe 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
Start with the transcriptome- combined use of tag for generic ChIP on chip
- maximize informational base to improve mouse experimentation- another use of ES cells to reduce animal experimentation
A generic multi-purpose allele design for tagging/functional analyses
Genetrap style targeting -- add FlEx
Knock-in of a protein tag in ES cells & miceTesta et al, (2003) Nature Biotechnology, 21, 443-447
Giuseppe TestaMisho Sarov
Problems of the (CaTZZ) TAP-Tag
1. The ZZ domain of protein A bind to endogenous IgG
2. Elution of the Calmodulin Binding domain from the beads
3. TEV is not the optimal protease
New double cassettesDevelopment of new Tag combinations
20 ng/l GST-TAPmouse brain extract 5mg/ml1:250 dilution (abundant protein)Coomassie blue stained gel
“In vitro” pull down
0%
50%
100%
STM E1 E2 B2
Mapping the mammalian core proteome- progress
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 Towards an optimized strategy
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
A. Shevchenko group:MPI-CBG, Dresden
Vineeth Surendranath*Patrice Waridel *Youri Kravatsky*
*supported by BMBF
Henrik ThomasShamil Sunyaev (Harvard Medical School, Boston)
David Drechsel(MPI-CBG, Dresden)
A. F. Stewart group:Technical University of Dresden Technical University of Dresden Genomics, BioZGenomics, BioZ
Senming Zhao*Michael Sarov *Daniel SchaftDaniel SchaftAssen RoguevAssen RoguevPim PijnappelPim PijnappelSandra LubitzStefan Glaser
*supported by BMBF
Frieder Schwenk(Artemis Pharmaceuticals, Koeln)
AcknowledgementsAcknowledgements
Open issues
recombineering strategy
the ideal protein tag
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
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
2. Obtain the BAC from the genome resources
3. Subclone according to design
4. Insert multi-purpose/conditional cassettes
EUCOMM dedicated software for targeting design