Yeast Pushes the Proteomic Envelope

NETWORKED: (A) Effects of the gal4D+gal perturbation are superimposed on a gene interaction network… Click for larger version (92K) Large-scale biology once conjured images of brute-force genetic screens resulting in collections of mutants. More recently, it has meant genome sequencing on unprecedented scales. But today, as this issue's Hot Papers demonstrate, the leading edge in large-scale biology is proteomics. As in the large-scale efforts of the past, researchers rely on simple model organisms to develop their methods. In the proteomics push, brewer's yeast, Saccharomyces cerevisiae, is creating yet another mold. "[It has been] a huge leg up to have [the yeast genome] available for the last seven years," says John Yates III, a professor at The Scripps Research Institute in La Jolla, Calif. "The genome itself is relatively simple ... and gene prediction is pretty accurate ... people have made reagents where every yeast protein is fused to other domains like GST [glutathione S-transferase] and GFP [green fluorescent protein] ... and now the deletion set is available." Yates says these benefits, and the sense of sharing and community in the field, are the reasons yeast leads the proteomics charge. Most of these Hot Papers1-4 have dramatically increased the number and kinds of proteins or protein interactions that are measured. A fifth study5 essentially put proteomics advances to use, describing the effects of various perturbations of a well-defined molecular pathway. Together, these papers expanded the knowledge of an important model organism and honed proteomic skills for application in higher eukaryotes. Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. M.P. Washburn et al., "Large-scale analysis of the yeast proteome by multidimensional protein identification technology," Nat Biotechnol, 19:242-7, 2001. (Cited in 236 papers) H. Zhu et al., "Global analysis of protein activities using proteome chips," Science, 293:2101-5, 2001. (Cited in 192 papers) Y. Ho et al., "Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry," Nature, 415:180-3, Jan. 10, 2002. (Cited in 247 papers) A.C. Gavin et al., "Functional organization of the yeast proteome by systematic analysis of protein complexes," Nature, 415:123-4, Jan. 10, 2002. (Cited in 301 papers) T. Ideker et al., "Integrated genomic and proteomic analyses of a systematically perturbed metabolic network," Science, 292:929-34, 2001. (Cited in 195 papers) MASSIVE MASS SPEC Michael Washburn1 described a technique charmingly called MudPIT (multidimensional protein identification technology), in which proteins pass directly from separation by liquid chromatography to detection by mass spectrometry. Yates, whose lab did the work, says the technology provides multiple advantages over older 2-D gel methods. "[We were] able to see fairly low-abundance proteins, [and by] using a different sample preparation method, we could see many more membrane proteins than people could see before," he says. Yeast researcher Mike Snyder of Yale University says, "There's no question that mass spectrometry is the way of the future ... this technology will replace 2-D gels in some form." The work allows for greater expansion into proteomics. "[The Yates paper] is the backdrop for doing many other experiments: looking at a large set of proteins under a lot of different conditions, or at protein modification in a global way," says Stan Fields, a professor at University of Washington, Seattle. Knowing that they can detect proteins for nearly all of an organism's proteome, researchers want to look at dynamic changes and modifications. "In that sense, it is a landmark paper in a field that is going to continue growing," says Fields. Heng Zhu and colleagues took an alternative approach to create a broad protein snapshot, developing the first whole proteome chip.2 "The dogma running around at the time was that you couldn't make protein chips," says Snyder, whose lab did the work. Doubts surrounded the ability to not only make the proteins, but also to keep them active while on a chip's surface. "[Zhu] showed you can lay down all 5,800 of these proteins, that they are still active," says Yates. Besides creating the chip, Zhu identified new proteins that interact with calmodulin, which has been widely studied, and with phospholipids, which are biologically important but are less well understood. "There were no good ways of finding all the phospholipid binding proteins before this," says Snyder. Adds Fields, "It is just the beginning of where protein chips are going to go." Chips continue to probe DNA and RNA binding, drug interactions, and more. Once the proteins and the chip are made, they become a resource for the entire yeast community. "Mike gives these things out," says Yates, referring to the proteins. As for the chips, Snyder and Zhu founded Branford, Conn.-based Protometrix to commercialize them. Two other papers published in the same issue of Nature describe large-scale, ultrasensitive mass spectrometry methods of identifying multiprotein complexes.3,4 Yuen Ho of MDS Proteomics, Toronto, Canada and academic and industry collaborators identified complexes using overexpressed proteins.3 Anne-Claude Gavin of Cellzome in Heidelberg, Germany and other European collaborators used proteins expressed at more natural levels.4 Despite some reports of high false-positive rates and a lack of concordance between the two datasets,6 the papers are still considered groundbreaking. "[Ho's and Gavin's work] were the first cases of using [this] technology on this kind of scale ... and together they have pushed the field forward," says Fields. PRETERNATURALLY PERTURBED Trey Ideker, an assistant professor at the University of California, San Diego, used a number of global technologies to systematically knock out an extremely well studied pathway, galactose metabolism, to refine the existing model.5 "[Ideker's paper] shows a way of applying global technologies to uncover new things that hadn't been seen before," says Snyder. Beginning with the known pathway for galactose metabolism, Ideker and colleagues knocked out key components and measured global changes in gene expression with mRNA microarrays. And, because mRNA and protein levels can differ, they also used isotope-coded affinity tagging and tandem mass spectrometry to measure protein levels in a subset of proteins that included other known galactose pathway components. In general, their findings agreed with the known pathway, but enough surprises popped up to make it interesting. For example, the gal10 gene resides near the pathway's end in the known model, and Ideker expected that disrupting it would not have far-reaching effects. "But that wasn't what happened. Every other gal gene [in the pathway] changed," he says. Based on the results, they tested new hypotheses and made changes to the current thinking of how the pathway components interact. Although it might be surprising that new information could come from experiments on such well-defined pathways, Ideker says the definition was incomplete: "People haven't been systematic about [experiments] because the technology wasn't available until recently." Speaking about the direction in which proteomics research is headed, both now and in the near future, some researchers say that they expect these kinds of global studies to continue revealing new information about old pathways. "I think we will see more of that," says Snyder. In addition, they expect a lot of comparative protein profiling and large-scale studies of protein-protein interactions. For example, the new technologies highlighted in these Hot Papers have recently been used to look at how protein levels change during the different stages of the malarial life cycle,7 and to show that a mitochondrial protein is transcribed from the antisense strand of the ribosomal RNA.8 The researchers also agree that yeast experiments provide a blueprint for proteomics in human tissues and cell lines. "The complication is deciding what you call the human proteome, which potentially could be millions of different isoforms," says Snyder. But, he says, a good start would be to "have one major isoform for every human gene." Ideker says that once a wiring diagram is designed to show how human cells work, pharmaceutical companies can run simulations, as Ford or Boeing does for product development. "Simulating a cell is a lot harder than [replicating] a car or a plane, but that is where production of drugs is going." Mignon Fogarty (mignon@welltopia.com) is a freelance writer in Santa Cruz, Calif. References 1. M.P. Washburn et al., "Large-scale analysis of the yeast proteome by multidimensional protein identification technology," Nat Biotechnol, 19:242-7, 2001. (Cited in 236 papers) 2. H. Zhu et al., "Global analysis of protein activities using proteome chips," Science, 293:2101-5, 2001. (Cited in 192 papers) 3. Y. Ho et al., "Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry," Nature, 415:180-3, Jan. 10, 2002. (Cited in 247 papers) 4. A.C. Gavin et al., "Functional organization of the yeast proteome by systematic analysis of protein complexes," Nature, 415:123-4, Jan. 10, 2002. (Cited in 301 papers) 5. T. Ideker et al., "Integrated genomic and proteomic analyses of a systematically perturbed metabolic network," Science, 292:929-34, 2001. (Cited in 195 papers) 6. C. von Mering et al, "Comparative assessment of large-scale data sets of protein-protein interactions," Nature, 417:399-403, 2002. 7. L. Florens et al., "A proteomic view of the Plasmodium falciparum life cycle," Nature, 419:520-6, 2002. 8. P.S. Coelho et al., "A novel mitochondrial protein, Tar1p, is encoded on the antisense strand of the nuclear 25S rDNA," Genes Dev, 16:2755-60, 2002. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Yeast Pushes the Proteomic Envelope

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	NETWORKED: (A) Effects of the gal4D+gal perturbation are superimposed on a gene interaction network… Click for larger version (92K)