TAPping the Power of Proteomics

The Faculty of 1000 is a Web-based literature awareness tool published by BioMed Central. It provides a continuously updated insider's guide to the most important peer-reviewed papers within a range of research fields, based on the recommendations of a faculty of more than 1,400 leading researchers. Each issue, The Scientist publishes a list of the 10 top-rated papers from a specific subject area, as well as a short review of one or more of the listed papers. We also publish a selection of comments on interesting recent papers from the Faculty of 1000's output. For more information visit www.facultyof1000.com. Most laboratory work is, by necessity, reductionist. A minute aspect of an organism, isolated from its contextual surroundings, is examined in excruciating detail, and the knowledge gained is pieced together with similar studies to gain a picture of the larger puzzle. A certain kinase is examined, for example, to see how well it will add a phosphate to a carefully selected substrate in a test tube. In this way, researchers gain a profound understanding of much of the living world. F1000 Top 10 All of Biology 1. A.C. Gavin et al., "Functional organization of the yeast proteome by systematic analysis of protein complexes," Nature, 415:141-7, Jan. 10, 2002. 2. A.R. Gendall et al., "The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis," Cell, 107:525-35, November 2001. 3. S.D. Tyner et al., "p53 mutant mice that display early ageing-associated phenotypes," Nature, 415:45-53, Jan. 3, 2002. 4. P. Nash et al., "Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication," Nature, 414:514-21, Nov. 29, 2001. 5. S. El-Din El-Assal et al., "A QTL for flowering time in Arabidopsis reveals a novel allele of CRY2," Nature Genetics, 29:435-40, Dec. 1, 2001. 6. I.E. Gallouzi, J.A. Steitz, "Delineation of mRNA export pathways by the use of cell-permeable peptides," Science, 294:1895-901, Nov. 30, 2001. 7. R.C. Robinson, "Crystal structure of arp2/3 complex," Science, 294:1679-84, Nov. 23, 2001. 8. W.M. Gray et al., "Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins," Nature, 414:271-6, Nov. 15, 2001. 9. D.S. Campbell et al., "Chemo-tropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation," Neuron, 32:1013-26, December 2001. 10. O. Stemmann et al., "Dual inhibition of sister chromatid separation at metaphase," Cell, 107:715-26, December 2001.Compiled Jan. 30, 2002 But the biological world, especially at the sub-cellular level, does not exist merely as discrete units or even pairs, contributing nothing to the whole but the sum of their parts. Those parts interact with each other to display properties not found when taken in isolation. Witness the choreography involved as the transcriptional or translational machineries create RNA or proteins, let alone their relationships to each other. The burgeoning field of proteomics has identified a huge number of cellular proteins. Techniques such as the two-hybrid system have indicated many pair-wise interactions among them. But that is merely "an inventory," says Anne-Claude Gavin. She and 37 of her colleagues at Cellzome took a huge step toward ascertaining the integration of cellular components in a paper that topped the Faculty of 1000's "All of Biology" top 10 list for Jan. 30.1 The researchers fished out more than 200 large protein complexes, each ranging in size from 2 to 83 components, from the baker's yeast, Saccharomyces cerevisiae. This tour de force was the company's first project, which Gavin says was undertaken as a "proof of concept" of its ability to harness this technology's power. "Our goal is to understand how an entire proteome is organized into functional units," she says. More than 80% of the proteins screened in this work were part of multiprotein complexes; most were interconnected through shared components. From this data, the researchers reconstructed a network of complexes. "It's the first time we can draw a map at that level of organization," notes Gavin. In doing so, they predicted the function of many previously unknown or unannotated proteins, principally through homology and "guilt by association," as the paper phrased it. The key to the work is tandem affinity purification (TAP), a biphasic method developed at the European Molecular Biology Laboratory and first published in 1999.2 (Several EMBL scientists went on to form Cellzome the following year.) A cassette encoding a TAP tag, consisting of a calmodulin- binding peptide, a specific enzyme cleavage domain, and Protein A from Staphylococcus aureus, was inserted next to a gene of interest. The resulting fusion proteins, along with whatever proteins associate with them, can be isolated from the cell lysates by their tag under mild conditions. In this case, the Cellzome researchers produced a library of 1,548 TAP-tagged yeast strains through homologous recombination. Tagged protein complexes are recovered by affinity selection on an IgG matrix, released by proteolytic cleavage, and repurified on calmodulin-coated beads. After elution, the proteins are analyzed using existing mass spectrometry (MS) techniques such as MALDI-TOF or LC/MS/MS (for mammalian purifications). TAP/MS is to be the basic platform for Cellzome, Gavin says. Their ultimate aim is to create drug targets by investigating the multifaceted relationships in the human proteome. Josh P. Roberts (tcwriter@msn.com) is a freelance writer in Minneapolis, MN. References 1. A.C. Gavin et al., "Functional organization of the yeast proteome by systematic analysis of protein complexes," Nature, 415:141-7, Jan. 10, 2002. 2. G. Rigaut, et al., "A generic protein purification method for protein complex characterization and proteome exploration," Nature Biotechnology, 17:1030-2, 1999.

TAPping the Power of Proteomics

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