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

Abstract

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 pH range of 3-10 with a relative molecular weight of 21-70 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, cDNA 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 after-cataracts.

Keywords: capsular block syndrome; liquefied after-cataract; liquid chromatography-tandem mass spectrometry.

Figure 1. Slit-lamp photograph showing fibrosis of CCC, anterior capsular opacity, a backward extension of the posterior capsule, and the presence of milky-white fluid between IOL and the posterior capsule.

Figure 2. Scheimplug photography of the anterior segment of the eye showing normal anterior chamber depth and relative density of the milky white substance behind the IOL.

Figure 3. A 27-gauge needle was inserted through the edge of the CCC into the capsular bag to extract aqueous humor from the anterior chamber along with the milky white substance.

Figure 4. Two-dimensional electrophoresis of normal lenses (A) and liquefied after-cataract (B)

1: Beta-crystallin B; 2: Glyceraldehyde-3-phosphate dehydrogenase; 3: Carbonyl reductase (NADPH) 1; 4: cDNA FLJ55253; 5: Gamma-crystallin D; 6: GAS2-like protein 3; 7: Sorbitol dehydrogenase; 8: cDNA FLJ60282; 9: Phosphoglycerate kinase; 10: Filensin.