washburn_MUDPIT_NAT_BIOTECH - 2001 Nature Publishing Group...

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nature biotechnology VOLUME 19 MARCH 2001 http://biotech.nature.com R ESEARCH A RTICLE 242 Large-scale analysis of the yeast proteome by multidimensional protein identification technology Michael P. Washburn 1† , Dirk Wolters 1† , and John R.Yates III 1,2 * We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liq- uid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analy- sis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low- abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins. Modern biologists can now observe quantitative changes in the expression levels of thousands of messenger RNA (mRNA) tran- scripts to determine the effects of a wide variety of perturbations to a cell 1 . However, there exists conflicting evidence regarding the corre- lation between mRNA and protein abundance levels 2–5 . Recent math- ematical modeling studies have demonstrated the need to know both the mRNA and protein expression levels of genes in order to describe a gene network 6,7 . The need to complement mRNA expression analy- sis has resulted in the emergence of the field of proteomics to direct- ly analyze protein expression levels from an organism. The analysis of a proteome requires the resolution of the proteins in a sample followed by the identification of the resolved proteins. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by mass spectrometry (MS) is the most widely used method of protein resolution and identification 8–10 . In 2D-PAGE, proteins are separated in one dimension by isoelectric point (pI) and in the other dimension by molecular weight (MW). High-throughput analysis of proteomes remains challenging because the individual extraction, digestion, and analysis of each spot from 2D-PAGE is a tedious and time-consuming process. As a result, the largest 2D-PAGE-based proteomic study to date identified 502 unique proteins for the Haemophilus influenzae proteome 11 . Portions of proteomes such as proteins with extremes in pI and molecular weight 12,13 , low-abundance proteins 14–16 , and membrane-associated or bound proteins 17,18 are rarely seen in a 2D-PAGE study. While efforts to alle- viate the current shortcomings in 2D-PAGE continue, we are explor-
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washburn_MUDPIT_NAT_BIOTECH - 2001 Nature Publishing Group...

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