Analysis of proteomic differences between liquefied aftercataracts and normal lenses using two-dimensional gel electrophoresis and mass spectrometry

AIM：To analyze and identify the proteomic differences between liquefied after-cataracts and normal lenses by means of liquefied chromatography-tandem mass spectrometry（LC-MS/MS）.METHODS：Three normal lenses and three liquefied after-cataracts were exposed to depolymerizing reagents to extract the total proteins.Protein concentrations were separated using two-dimensional gel electrophoresis（2-DE）.The digitized images obtained with a GS-800 scanner were then analyzed with PDQuest7.0 software to detect the differentially-expressed protein spots.These protein spots were cut from the gel using a proteome work spot cutter and subjected to in-gel digestion with trypsin.The digested peptide separation was conducted by LC-MS/MS.RESULTS：The 2-DE maps showed that lens proteins were in a p H range of 3-10 with a relative molecular weight of21-70 kD.The relative molecular weight of the more abundant proteins was localized at 25-50 kD,and the isoelectric points were found to lie between PI 4-9.The maps also showed that the protein level within the liquefied after-cataracts was at 29 points and significantly lower than in normal lenses.The 29 points were identified by LC-MS/MS,and ten of these proteins were identified by mass spectrometry and database queries：beta-crystallin B1,glyceraldehyde-3-phosphate dehydrogenase,carbonyl reductase（NADPH）1,c DNA FLJ55253,gamma-crystallin D,GAS2-like protein 3,sorbitol dehydrogenase,DNA FLJ60282,phosphoglycerate kinase,and filensin.CONCLUSION：The level of the ten proteins may play an important role in the development of liquefied aftercataracts.