INTERLABORATORY REPRODUCIBILITY OF YEAST PROTEIN-PATTERNS ANALYZED BYIMMOBILIZED PH GRADIENT 2-DIMENSIONAL GEL-ELECTROPHORESIS

An interlaboratory comparison was conducted on the positional and quan titative reproducibility of yeast proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) using isoelectric focus ing with immobilized pH gradient (pH 4-7) in the first dimension. The basic experimental setup was as follows: one laboratory prepared and d istributed a [S-35]methionine-labeled total yeast protein extract (Got eborg, Sweden), another laboratory prepared the IPG strips to be used by all labs in this study (Munich, Germany), the third laboratory (Aar hus, Denmark) circulated the protocols and coordinated the modest atte mpts to unify them. Samples were run horizontally in the first dimensi on and vertically in the second. The gels were sent to Goteborg for pr ocessing by phosphoimager technology and computerized image analysis ( PDQuest), and the 2-D PAGE resolved proteins were located and quantifi ed automatically. A subset of 470 spots was manually matched in all ge ls out of an average of 1328 resolved proteins. The positional interla boratory comparison revealed great pattern reproducibility, the correl ation coefficient in no case being less than 0.9994. In absolute terms an average deviation of 2.8 mm (x-position) and 1.8 mm (y-position) w ere obtained for all nine gels (three gels per lab). The interlaborato ry comparison of protein quantitation displayed higher variability, an d the best correlation coefficient generated was 0.975. An average sta ndard deviation of 34.5% was calculated for protein quantitation inclu ding all three labs, a value slightly higher than the intralaboratory variation (range 20-28%) Thus, despite differences in protocols, chemi cals and equipment, the immobilized pH gradient technology gave extrem ely high positional and quantitative reproducibility. This will greatl y facilitate the exchange of data and the establishment of multi-user image-based 2-D gel databases.