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