The proteome research of human lens epithelial cells by two-dimensional gel electrophoresis and mass spectrometry

AIM: To establish the techniques in the proteome research of human lens epithelial cells,including the techniques of two-dimensional electrophoresis and mass spectrometry.METHODS: Total protein of cultured human lens epithelial cells was extracted with two kinds of different methods.The proteins were separated using immobilized pH gradients 2-DE and visualized by silver staining.The digitized images obtained by GS-800 scaner were then analyzed with PDQuest software in order to establish the differential expression profiles.The differential expressed protein spots were cut from the gels using proteomework spot cutter and subjected to in-gel digestion with trypsin.The digested peptide separation was conducted by a Finnigan LCQ MS coupled with a Surveyor HPLC system. RESULTS: A high resolution and reproducible 2-ED image was successfully obtained.The maps of 2-DE showed that lens proteins were in the section of pH 4-7 which the relative molecular weight was 17-72 kD.Relative molecular weight of more abundant proteins was localized at 19-50 kD,as well as the isoelectric points were found to lie between PI 5-7.Two of these proteins were identified by mass spectrometry and database queries.CONCLUSION: A stable protein maps of human lens epithelial cells is constructed.The technique will be used in human lens research to characterize physiological processes and diseases.