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The goal of proteomics is a comprehensive, quantitative description of protein expression and its changes under the influence of biological perturbations such as disease or drug treatment. Quantitative analysis of protein expression data obtained by high-throughput methods has led us to define the concept of “regulatory homology” and use it to begin to elucidate the basic structure of gene expression control in vivo. Such investigations lay the groundwork for construction of comprehensive databases of mechanisms (cataloguing possible biological outcomes), the next logical step after the soon to be completed cataloguing of genes and gene products. Mechanism databases provide a roadmap towards effective therapeutic intervention that is more direct than that offered by conventional genomics approaches.

We have improved upon the reference two-dimensional (2-D) electrophoretic map of rat liver proteins originally published in 1991 (N. L. Anderson et al., Electrophoresis 1991, 12, 907-930). A total of 53 proteins (102 spots) are now identified, many by microsequencing. In most cases, spots cut from wet, Coomassie Blue stained 2-D gels were submitted to internal tryptic digestion [2], and individual peptides, separated by high-performance liquid chromatography (HPLC), were sequenced using a Perkin-Elmer 477A sequenator. Additional spots were identified using specific antibodies.

An updated two-dimensional electrophoretic map of human plasma proteins is presented, together with a complete listing of the individual protein spots, their locations, size and isoelectric points relative to internal charge standards. Forty-nine polypeptide species are identified, many consisting of multiple spots differing in glycosylation or sequence (e.g., immunoglobulins). A further series of 35 as yet uncharacterized proteins is indicated.

A standard two-dimensional (2-D) protein map of Fischer 344 rat liver (F344MST3) is presented, with a tabular listing of more than 1200 protein species. Sodium dodecyl sulfate (SDS) molecular mass and isoelectric point have been established, based on positions of numerous internal standards. This map has been used to connect and compare hundreds of 2-D gels of rat liver samples from a variety of studies, and forms the nucleus of an expanding database describing rat liver proteins and their regulation by various drugs and toxic agents. An example of such a study, involving regulation of cholesterol synthesis by cholesterol-lowering drugs and a high-cholesterol diet, is presented. Since the map has been obtained with a widely used and highly reproducible 2-D gel system (the Iso-Dalt system), it can be directly related to an expanding body of work in other laboratories.

Liver proteins of male C57BL/6T mice treated with 0, 50, or 250 mg/kg Aroclor 1254 were analyzed by high-resolution two-dimensional (2-D) electrophoresis. The resulting patterns were processed using a computerized image analysis system and quantitative data selected for a total of 150 protein spots. On the basis of an analysis of liver proteins form five animals in each treatment group, we found 31 proteins that showed quantitative differences attributable to treatment with chlorinated hydrocarbons at a high level of statistical significance. One of the altered proteins appears to be Mitcon:2, a heat-shock sensitive mitochondrial matrix polypeptide; another appears likely to be microsomal cytochrome b5. The results indicate that quantitative 2-D protein mapping may reveal much more detail regarding the in vivo effects of toxic xenobiotics than has previously been available, and thus allow a more informative approach to the testing of toxic compounds.

A simple and versatile optical technique for viewing discontinuities in the thickness of horizontal isoelectric focusing gels is described. The discontinuities in the gel are reproducible and relate directly to the ampholyte distribution. Characterization of the capture of the discontinuities in the gel is presented as well as a demonstration of how they may used for localization of focused proteins.

Using the ISO-DALT system for two-dimensional (2-D) electrophoresis and the TYCHO system for computer analysis of the resulting protein maps, we obtained high quality quantitative protein abundance data from Coomassie Brilliant Bluestained gels of mouse liver samples. High resolution gels allow more than 100 proteins to be measured with coefficients of variation less than 15 %. A comparison of results from two mouse strains (C57BL/6 and BALB/c) and the cross between them (BCF1) shows that a large number of qutive polymorphisms can be detected, and that, as expected, the amount of protein produced in the heterozygote is intermediate between the parental values. The system described is shown to be capable of reliably detecting decreases in protein abundance such as those expected to result from radiation-induced deletion of one copy of a gene. The implications of these results for the study of gene regulation are discussed in relation to applications in genetics, toxicology, and differentiation.

A method of testing batches of ampholytes is presented. By using carbamylated charges standards to co-electrophorese with the protein sample in the first-dimension isoelectric focusing gel, one can monitor, after running and staining the second-dimension sodium dodecyl sulfate (SDS) slabs gel, the continuity of the pH gradient. Charge standards can also be used to check the reproducibility of the pH gradient among batches of ampholytes and to modify the new batch with a small amount of a narrow range ampholyte to assure reproducibility of experiments. Ampholytes for comparison were obtained from three major manufacturers.

Human plasma proteins separated by high-resolution two-dimensional electrophoresis have been electrophoretically transferred to sheets of nitrocellulose using a modification of the method of Towbin, Staehelin, and Gordon [8]. Although the proteins have been denatured in sodium dodecyl sulfate and separated into subunits, the nitrocellulose-bound molecules still react with appropriate specific antisera even after storage of the transfer in air at room temperature for 5 months. Of 25 proteins whose location in the pattern had been previously determined, 24 are specifically revealed on transfers of whole plasma patterns by appropriate antiserum. In addition, 6 previously unidentified proteins (prothrombin, C1s, C4γ, C1s inhibitor, Ig J-chain, and a1AP glycoprotein) have been identified in the pattern for the first time using the transfer technique. It therefore seems likely that a large majority of proteins (> 96 % in this study) retain sufficient conformation throughout the analytical procedure (or can regain it easily afterwards) to be recognized immunologically. The transfer technique thus constitutes a generally useful immunological “third-dimension” in the high-resolution separation of proteins. Of three monoclonal antibodies similarly tested, none could detect antigen transferred to nitrocellulose from a two-dimensional gel, while each bound specifically to the appropriate antigen absorbed in native form to the nitrocellulose.

Procedures to measure two important properties of two-dimensional electrophoresis systems have been developed. These procedures evaluate the overall resolution and positional reproducibility of two-dimensional gel patterns. Using these measures we show that various state of the art gel systems can produce patterns exhibiting resolution measures of 15 000–50 000 (yielding practical upper bounds of about 2000–6000 detectable proteins in typical cellular samples) and that positional accuracy for most spots in gel-to-gel comparisons is better than a spot width (0.5–2.0 mm). Implemented as part of a computerized image analysis system for two-dimensional gel data, these two measures can be used in the selection and optimization of gel systems, and in the selection of sets of well-resolved, well-matched spots for genetic and other quantitative sudies.