Preliminary proteome analysis of mouse embryonic fibroblast conditioned medium

Objective To perform the proteome analysis of conditioned medium prepared from mouse embryonic fibroblast feeder layers by 2-dimensional (2D) electrophoresis and mass spectrometry and to find out the possible differentiation-inhibitory factor in conditioned medium. Methods Feeder layers were prepared by 60Co γ-irradiation on mouse embryonic fibroblast. Insulin-transferrin-sodium selenite supplemented medium was used to culture the feeder layers for 24 hours. The condioned medium prepared from mouse embryonic fibroblast feeder layers were made into powder by lyophilization, the re-dissolved solution was applied to Sephadex G-50 gel filtration chromatography, and then cold acetone was used to precipitate the proteins in the eluted solution. The protein samples were applied to 2D electrophoresis. The 2D images were analyzed by 2D image analysis software. Selected protein spots were digested by trypsin, analyzed by mass spectrometry, and then searched against the NCBInr batabase using Mascot MS/MS Ions Search. Results The protein samples extracted from mouse embryonic fibroblast feeder layersconditioned medium could be used for 2D electrophoresis. On 2D images, there were (221±67) spots. Most of the proteins were located in the region of MW 20~70 kD,pI 4~8. Using mass spectrometry, we preliminarily identified 13 spots: 3 keratins, 3 transferrins, 1 trypsin precursor, 2 unknown proteins (3 spots), 1 connexin 46, 1 beta-galactoside binding protein, and 1 secreted protein, acidic and rich in cysteine. Conclusion Conditioned medium prepared from mouse embryonic fibroblast feeder layers contain beta-galactoside binding protein and secreted protein, acidic and rich in cysteine.