Transcript Tandem Affinity Purification Tag

Functional organization of the yeast
proteome by systematic analysis of
protein complexes
Anne-Claude Gavin,
Gavin Markus
et al. Bosche, Roland Krause, Paola Grandi, Martina
Marzioch, Andreas Bauer, Jorg Schultz, Jens M. Rick, Anne-Marie Michon,
Cristina-Maria Cruciat, Marita Remor, Christian Hofert, Malgorzata
Schelder, Miro Brajenovic, Heins Ruffner, Alejandro Merino, Karin Klein,
Manuela Hudak, David Dickson, Tatjana Rudi, Volker Gnau, Angela Bauch,
Sonja Bastuck, Bettina Huhse, Christina Leutwein, Marie-Anne Heurtier,
Richard R. Copley, Angela Edelmann, Erich Querfurth, Vladimir Rybin,
Gerard Drewes, Manfred Raida, Tewis Bouwmesster, Peer Bork, Bertrand
Seraphin, Bernhard Kuster, Gitte Neubauer, and Giulio Superti-Furga
(Nature 415. 2002)
Background Information
1. Proteins rarely act alone
2. Comprehensive protein interaction studies thus far:
• two-hybrid systems (ex vivo)
• protein chips (in vitro)
• GST pull-downs (in vivo)
3. Authors used tandem-affinity purification (TAP)
and mass spectrometry
Rationale for Using TAP/MS Method
1. Fast purification with high yield
2. In vivo
3. Over-expression of proteins not required
4. Prior knowledge of protein complex not required
5. Purified complex can be used in a variety of studies
The Tandem Affinity Purification Tag
target protein
target protein
TAP Purification Strategy
TAP Purification Strategy
TAP Purification Strategy
TAP Purification Strategy … in detail
1. PCR of the TAP cassette
2. Transformation of yeast cells
(homologous recombination)
3. Selection of positive clones
4. Large-scale cultivation
5. Cell lysis
Tandem affinity purification
6. One-dimensional SDS-PAGE
7. MALDI-TOF protein identification
8. Bioinformatic data interpretation
The Experiment
Processed
1,739 genes (1,143 human orthologues)
Purified
589 protein assemblies
Annotated 232 multiprotein complexes (98 known, 134 new)
Proposed new/additional cellular roles for 344
proteins (231 new)
Identified 1,440 distinct gene products
The Experiment …cont’d
 9% of the 232 TAP complexes had no new
component
 2-83 components per TAP complex
 Assigned cellular roles to complexes according to
YPD and literature studies
 Nine functional categories
Some Statistics
A Higher-Order Map
http://yeast.cellzome.com
Linked Complexes
Results of the Experiment
• Orthologues tend to interact with orthologues
(53% vs
31%)
• Essential genes tend to interact with essential genes
(44% vs 17%)
• Existence of “orthologous proteome” for eukaryotes?
Some Faults of the TAP Method
• “Super” unstable protein complexes
• TAP tag may interfere with complex
• Transient interactions
Low stoichiometric complexes
Physiology-specific interactions
Conclusions
•This TAP/MS method is the largest analysis of protein
complexes
•Allows efficient identification of low-abundance
proteins
•Allows purification of very large complexes
•Can complement other biochemical techniques
•Lower-order maps and higher-order maps provide
crucial information
Great Summer Reading
Rigaut, G. et al. A generic protein purification method for protein
complex characterization and proteome exploration. Nature
Biotechnol. 17, 1030-1032 (1999).
Puig, O. et al. The tandem affinity purification (tap) method: a
general procedure of protien complex purification. Methods 24, 218229 (2001).