1. Functional organization of the yeast proteome by systematic analysis of protein complexes
Anne-Claude Gavin, Markus 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 )
Anne-Claude Gavin et al.

2. Background Information
Proteins rarely act alone
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
Slide 2
At the biochemical level, proteins rarely act alone, but rather interact with one another, sometimes in complexes, to carry out specific cellular processes.
Two hybrid systems: (+) pairwise/binary interactions between proteins; transient associations
(-) characterization of protein complexes
Protein chips: (-) it is in vitro
TAP: The authors used a technique called tandem-affinity purification coupled with mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharyomeces cerevisiae, the budding or baker’s yeast.

3. Rationale for Using TAP/MS Method
Fast purification with high yield
In vivo
Over-expression of proteins not required
Prior knowledge of protein complex not required
Purified complex can be used in a variety of studies
Slide 3
Allows for fast purification with high yield of protein complexes under native, physiological conditions; therefore, this is an in vivo approach. In addition, the proteins under study do not have to be overexpressed, in fact it is preferred to maintain the expression of the fusion, target protein at natural levels. This is because protein overexpression can result in nonspecific or unnatural protein interactions. Prior knowledge of complex composition and function is not necessary. Since the purification procedure is gentle, the purified complex can be used protein identification, functional, or structural studies.

4. The Tandem Affinity Purification Tag
target protein
target protein

5. TAP Purification Strategy

6. TAP Purification Strategy

7. TAP Purification Strategy

8. TAP Purification Strategy … in detail
PCR of the TAP cassette
Transformation of yeast cells (homologous recombination)
Selection of positive clones
Large-scale cultivation
Cell lysis Tandem affinity purification
One-dimensional SDS-PAGE
MALDI-TOF protein identification
Bioinformatic data interpretation

9. The Experiment Processed 1,739 genes (1,143 human orthologues)
Purified protein assemblies
Annotated 232 multiprotein complexes (98 known, 134 new)
Proposed new/additional cellular roles for proteins (231 new)
Identified 1,440 distinct gene products

10. 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
Slide 10
Of all 232 TAP complexes, only 9% had no new component. The size of the TAP complexes ranged from 2-83 components, with an average of 12 components per complex. Assigned cellular roles to complexes by computing functional assignments of the individual components according to YPD and by literature searching.

11. Some Statistics

12. A Higher-Order Map http://yeast.cellzome.com Slide 12
After the individual proteins of the complexes have been identified, the authors want to study the relationships between complexes to understand the integration and coordination of cellular processes.
This is a protein complex network, it provides a functional description of the eukaryotic proteome at a higher level of organization. The complexes are linked to each other if they share components. The cellular roles of the complexes are color coded. Dark blue = transcription, DNA maintenance, chromatin structure. Violet = intermediate and energy metabolism.
The website has a software package that allows you to navigate through this proteome map at both the complex and protein levels.

13. Linked Complexes Slide 13
This is an example of a complex linked to two other complexes by virtue of shared components. This illustrates the relationship between the protein and complex levels of organization.

14. 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?
Slide 14
Orthologous proteins tend to interact with complexes enriched with other orthologues (mean 53%). Nonorthologous proteins have a lower tendency for such interactions (31%). The same goes for the interaction of essential genes. This suggests the existence of an “orthologous proteome” that may represent core functions for the eukaryotic cell.

15. Some Faults of the TAP Method
“Super” unstable protein complexes
TAP tag may interfere with complex
Transient interactions
Low stoichiometric complexes
Physiology-specific interactions
Slide 15
The TAP method may not be able to detect complexes where proteins do not interact with sufficient stability. The TAP tag may interfere with complex formation or protein localization and function. This method may also fail to detect transient interactions, low stoichiometric complexes, and those interactions occurring in specific physiological states which exponential growing cells do not exhibit.

16. 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
Slide 16
Lower-order maps and higher-order maps provide crucial information for drug discovery efforts and may aid in the choice and evaluation of drug targets.

17. Great Summer Reading
Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotechnol. 17, (1999).
Puig, O. et al. The tandem affinity purification (tap) method: a general procedure of protien complex purification. Methods 24, (2001).