A sampling of the yeast proteome.

In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome.

Proteome studies of Saccharomyces cerevisiae: identification and characterization of abundant proteins.

Two-dimensional (2-D) gel electrophoresis can now be coupled with protein identification techniques and genome sequence information for direct detection, identification, and characterization of large numbers of proteins from microbial organisms. 2-D electrophoresis, and new protein identification techniques such as amino acid composition, are proteome research techniques in that they allow direct characterization of many proteins at the same time. Another new tool important for yeast proteome research is the Yeast Protein Database (YPD), which provides the sequence-derived protein properties needed for spot identification and tabulations of the currently known properties of the yeast proteins. Studies presented here extend the yeast 2-D protein map to 169 identified spots based upon the recent completion of the yeast genome sequence, and they show that methods of spot identification based on predicted isoelectric point, predicted molecular mass, and determination of partial amino acid composition from radiolabeled gels are powerful enough for the identification of at least 80% of the spots representing abundant proteins. Comparison of proteins predicted by YPD to be detectable on 2-D gels based on calculated molecular mass, isoelectric point and codon bias (a predictor of abundance) with proteins identified in this study suggests that many glycoproteins and integral membrane proteins are missing from the 2-D gel patterns. Using the 2-D gel map and the information available in YDP, 2-D gel experiments were analyzed to characterize the yeast proteins associated with: (i) an environmental change (heat shock), (ii) a temperature-sensitive mutation (the prp2 mRNA splicing mutant), (iii) a mutation affecting post-translational modification (N-terminal acetylation), and (iv) a purified subcellular fraction (the ribosomal proteins). The methods used here should allow future extension of these studies to many more proteins of the yeast proteome.

A Saccharomyces cerevisiae Internet protein resource now available.

The QUEST Protein Database Center is now making available two Saccharomyces cerevisiae protein databases via the Internet. The yeast electrophoretic protein database (YEPD) is a database of approximately one hundred protein identifications on two-dimensional gels. The yeast protein database (YPD) is a database of gene names and properties of over 3500 yeast proteins of known sequence. These databases can be accessed via a World-Wide Web (WWW) server (URL http:@siva.cshl.org). YPD is available via public ftp (isis.cshl.org) as well, in a spreadsheet format, and in ASCII format. When accessed via WWW, both of these databases have hypertext links to other biological data, such as the SWISS-PROT protein sequence database and the Saccharomyces Genome Database (SacchDB), and to each other.

Reduced numatrin/B23/nucleophosmin labeling in apoptotic Jurkat T-lymphoblasts.

Jurkat T-lymphoblasts were induced to undergo apoptosis by treatment with either EGTA (5 mM/24 h) or a high concentration of lovastatin (100 microM/48 h) to identify proteins that exhibited coordinate regulation between the two treatments and thus provide candidate proteins in the common apoptotic induction pathway. A pure population of apoptotic cells, as determined by morphology, "DNA laddering," and flow cytometry, was obtained by Percoll density gradient centrifugation. Cells of increased buoyant density were clearly apoptotic by all criteria. Following this gradient centrifugation, the cells were labeled with [35S]methionine/cysteine, and lysates were separated by two-dimensional polyacrylamide gel electrophoresis. Surprisingly, the two-dimensional polyacrylamide gel electrophoresis patterns generated from the apoptotic cells did not differ dramatically from that of control cells. Thus, apoptotic Jurkat cells are able to synthesize new proteins and do not exhibit extensive proteolysis. Subsequent quantitative analysis revealed that only five proteins exhibited decreases in turnover that were common to the two treatments. No increases in protein turnover were able to be confirmed across the replicate experiments. One of the proteins that showed decreased labeling by both apoptotic inductions was an abundant nuclear protein with a pI of 5.1 and M(r) 40,000. This protein was identified as numatrin/B23/nucleophosmin (NPM) based on internal amino acid sequence, and this identity was confirmed by immunoblotting and mass spectrometry. NPM is implicated in a range of diverse cellular functions, but its role in apoptosis is unclear.

Protein identifications for a Saccharomyces cerevisiae protein database.

The rapid progress in understanding the genes of the yeast Saccharomyces cerevisiae can be supplemented by two-dimensional (2-D) gel studies to understand global patterns of protein synthesis, protein modification, and protein degradation. The first step in building a protein database for yeast is to identify many of the spots on 2-D gels. We are using protein sequencing, overexpression of genes on high-copy number plasmids, and amino acid analysis to identify the proteins from 2-D gels of yeast. The amino acid analysis technique involves labeling yeast samples with different amino acids and using quantitative image analysis to determine the relative amino acid abundances. The observed amino acid abundances are then searched against the current database of 2600 known yeast protein sequences. At present about 90 proteins on our yeast maps have been identified, and the number is rising rapidly. With many known proteins on the map, it will soon be possible to use 2-D gel analysis to study regulatory pathways in normal and mutant yeast, with knowledge of many the protein products that respond to each genetic or environmental manipulation.

REF52 on Global Gel Navigator: an internet-accessible two-dimensional gel electrophoresis database.

Until recently, the REF52 2-D gel database of experiments with rat cell lines was accessible only with special software. This database has now been made available to all investigators with access to the Internet, using the World Wide Web (WWW) technology. The package which delivers the database through the WWW has been named the Global Gel Navigator and can be used to explore the data by several methods, including the direct selection of proteins in the displayed gel using the mouse.

A distributed system for two-dimensional gel analysis.

The Quest II system is a new two-dimensional (2D) gel analysis software system for the construction and analysis of 2D gel protein databases. A new architectural approach to 2D gel software systems has been utilized. This architecture is based on a tightly coupled client/server model. There are three layers to the system architecture: (i) a database layer consisting of three database servers, (ii) a compute layer consisting of three compute servers and (iii) an extensible user interface layer currently consisting of user interface tools for linearization and merging of scanned images, the segmentation and detection of protein spots on the images, matching, editing, and analysis of gels. The ability to store and retrieve the large volume of spot data inherent in 2D gel analysis while utilizing database technology is demonstrated.

Evaluation of storage phosphor imaging for quantitative analysis of 2-D gels using the Quest II system.

The advent of storage phosphor technology has been of considerable benefit to the imaging of gel-separated radiolabeled proteins due to the rapid and quantitative nature of the data acquisition process. Previously, times over one month were required to obtain fluorographs of the same gel to yield data of sufficient dynamic range for quantitative analysis of high-resolution two-dimensional (2-D) gels. As we are in the process of building a human 2-D gel protein database, and therefore have a high throughput of 2-D gels both to image and quantitate using the Quest II software, we undertook an evaluation of a storage phosphor imager, including an evaluation of signal fade. The results of this evaluation demonstrate the feasibility of using such a system, and we describe the procedures that allow us to use this technique for quantitative analysis of many complex 2-D gel patterns. These procedures include a useful batch printing program that allows printing of many images in a non-interactive mode. Examples will be presented of how autoradiography, using storage phosphor plates and the Quest II system, have enabled us to begin building a human 2-D gel protein database including posttranslational modification information, without the previous time constraints associated with such a project.

Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli.

Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).

Age dependent changes in the expression of Drosophila mitochondrial proteins.

A subset of 43 [35S]methionine labeled mitochondrial proteins, identified on two dimensional electrophoresis gels of whole body extracts of adult Drosophila, were compared between two age groups. These flies, which except for their spermatocytes consist entirely of postmitotic cells, were 6 days old and 38 days old, with the mean life span of this inbred strain being 32.5 days. Qualitative changes in any of these mitochondrial polypeptides were not found, but significant quantitative differences were observed. Quantitation of the 43 mitochondrial proteins was carried out by computer assisted microdensitometry of autoradiograms of the gel patterns. The total amount of isotope incorporated into the mitochondrial proteins of the senescent flies decreased to 71% of that of the young insects. This decrease was heterogeneously distributed among the 43 proteins; however, six remained unchanged and one protein (mol. wt = 75; pI = 7.4) was increased 2.4 times in the senescent flies. These data clearly provide evidence that the age-dependent changes in the expression of mitochondrial proteins are quantitative and not qualitative.

Identification of polypeptides on two-dimensional electrophoresis gels by amino acid composition.

We present a method that can, in principle, provide tentative identification of a substantial proportion of the polypeptides resolvable on a given two-dimensional electrophoresis gel. Computerized microdensitometry of autoradiograms from 20 gels labeled in turn with each of the 20 common amino acids provides the data for simultaneously measuring the amino acid composition of all polypeptides of interest on the gel. These compositions are then compared with computer data bases of known protein compositions. Similarity between a known and an unknown polypeptide with comparable molecular mass indicates a potential identification, which can then be confirmed with conventional techniques. We illustrate this technique by applying it to the identification of proteins in a transformed human cell line (HuT-14).

Normalization and reproducibility of mass profiles in the detection of individual differences from urine.

The mass profiles of urine samples and their reproducibility (coefficient of variation) obtained by field-ionization mass spectrometry (volcano source) depend upon the choice of normalization method. We compared six different normalization techniques in terms of improved reproducibility and sensitivity. Several simple procedures markedly improved reproducibility while preserving the high degree of sensitivity. Although these procedures gave similar and satisfactory results, others were clearly inferior and are not recommended for clinical profiling.