Anal. Chem. 1983, 55,2345-2348
2345
Computerized Analysis of Two-Dimensional Electrophoretograms William R. Hruschka* and David R. Massie Instrumentation Research Laboratory, Beltsville Agricultural Research Center, U.S. Department of Agriculture, Beltsville, Maryland 20705 James D. Anderson Plant Hormone Laboratory, Beltsville Agricultural Research Center, U.S. Department of Agriculture, Beltsville, Maryland 20705
Autoradlographs of soybean proteins separated by two-dlmenslonal gel electrophoresis were made, and thelr optlcal denslty patterns were dlgltlzed wlth an In-house-deslgned flatbed densltometer. Fortran mlnlcomputer programs were developed that smoothed the dlgltlzed data, removed background, reproduced the original autoradlograph patterns, and quantlfled the lndlvldual protelns. Dlgltlratlon contrlbuted less than 2% error to quantlflcatlon, and gel-togel varlatlon was less than 15%. Both the dlgltlrer and mlnlcomputer provlde for a moderatacost automated procedure that should be e& peclally useful In laboratories producing electrophoretograms of radloactlvely labeled protelns at the rates of about 300 per year.
In 1975, O’Farrell demonstrated the resolving power of two-dimensional gel electrophoresis for the separation of proteins (I). The method separates proteins by charge in the first dimension and by mass in the second dimension. After electrophoresis, proteins are detected by staining or, if labeled with radioactivity, by autoradiography or fluorography. Over proteins can be clearly separated on one gel. When such numbers of proteins are separated, as they usually are in biological work, their quantification by automated procedures is almost a necessity. The first attempts a t developing automated quantification procedures were published in 1978 (2-4), and several laboratories are now in various stages of developing their own such procedures (5-20). The potential of a fully computerized system is outlined in ref 20, along with a short review of the field. In our study of the growth processes in germinating soybean seeds, we are using two-dimensional electrophoresis to examine the effects of various treatments on protein synthesis. Because of the large number of newly synthesized proteins during germination, we felt it necessary to computerize data acquisition and analysis. We report here on procedures we developed for digitizing, quantifying, and displaying data obtainable from autoradiographs of individual gel electrophoretograms. The low-cost digitizer we developed records optical densities directly and can be assembled from off-the-shelf components. Quantification and display are accomplished with a minicomputer, which should be of interest to researchers with limited computer facilities. The data handling is simpler than most reported, without noticeable loss of precision. This allows the speed of the computer program to be comparable to that of more elaborate programs run on bigger computers. Digitization and between-gel variations contributed errors of
