Proteomics Study In Human Vitreous Humor Of Proliferative Retinopathy

Objective (1)To establish and optimize the two-dimensional electrophoresis /Mass Spectrometry (2-DE/MS) technique for human vitreous humor (VH) proteomics. (2)Separated VH proteins of patients with proliferative vitreoretinopathy (PVR) and donor eyes (controls) with 2-DE, analyze and compared the VH 2-DE profiles to find out the significative discrepant proteins with PDQuest software. (3)Identified these proteins with MALDI-TOF-MS and search for the peptide mass fingerprints (PMFs), confirmed the disease-specific proteins (DSPs) of PVR in order to offer some foundational information about the diagnosis and medication of PVR.Methods (1) Total soluble proteins of VH sample were extracted from 14 human donor eyes (24hr post-mortem) and 21 eyes of patients with PVR from vitrectomy, harvest vitreous samples from the middle of the vitreous cavity. (2)For purify and concentrate the sample proteins, spallation and precipitation have been executed. Immobilized pH gradient (IPG) gels and even SDS polyacrylamide gels were selected for the electrophoresis, the gels were stained by silver nitrate after the electrophoresis were completed. Experimental conditions were adjusted to find out the best one for VH. (3) Scanned 2-DE gels and analysis the protein spots in the 2-DE profiles between two groups by PDQuest software to find out the significant discrepant protein spots. (4) Discrepant protein spots were extracted and been enzymolysis and progressing mass spectrogram identification, then according the results of PMFs to find the discrepant proteins by searching internet using Mascot and MS-fit software from SWISS-PORT database.Results (1) Compared two precipitation methods'effect of VH protein's purification, we find acetone precipitation is a compatible extracted method for 2-DE of VH protein. (2)By IPG(pH4-7, L) running isoelectric focusing in the first dimension and casting 12% SDS even gel in the second dimension, 2-DE maps of VH is obtained which were more than 400 spots distributing evenly, and the repeatability of the maps is satisfactied. (3)Compared the profiles of two groups we found 12 significative discrepant protein spots, which 9 of them were homo-expression and 3 of them were hyp-expression. (4) There are 4 proteins have been confirmed through the MS identify which is Guanine nucleotide-binding protein G, S100A7(homo-expression) and Phosducin-like protein, Sorting nexin 11 (hyp-expression).Conclusion (1) Establishment and optimization of the harvest methods for VH samples and 2-DE/MS technique for VH proteomics may be effectively applied in the study of VH proteome and are useful to further study. (2) Notable difference were observed between the 2-DE profiles of PVR group and control group, and 5 proteins were identified which 4 proteins may be the DSPs, namely G protein, S100A7, SNX11, PHLP. (3) The homo- or hyp-expression of these proteins are also associated with cell's differentiation and proliferation which may be play an important role in the progress of PVR to promote PVR cells'abnormal differentiation and proliferation.