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R Koyama, T Nakanishi, A Shimizu, T Ikeda; A Two-dimensional Electrophoreisis Map of Human Vitreous Proteins . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3498.
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Purpose: The analysis of soluble protein profile in the vitreous humor (VH)may elucidate the pathogenesis of various retinopathies especially those accompanied by blood vessel growth into the vitreous. In diseases such as retinopathy of prematurity and proliferative diabetic retinopathy production of angiogenic and antiangiogenic factorc by retinal cells may change, and consequently the concentration of these factors in the VH may also change. This study aims to make a catalogue of soluble protein in humor VH and to compared the expressed proteins in VH between diadetic retinopathy, macula hole . Method: We started first-dimensional isoelectric foucucing then proceeded SDS-PAGE. The images of stained gels were studied with ImageMaster 2D Elite software. Protein spots were excised and were alkylated with iodoacetoamide, digested with TPCK modified trypsin. The digests were extracted and then the extracs were concentrated, resolved with 0.1% trifluoroacetic acid. MALDI mass spectra were acquired on a Voyager DE-PRO MALDI-TOEMS mass spectorometer (PE Biosystems, Framingham, MS) equipped with a delayed extraction source and a 337 nm pulsed nitrogen laser. MS/MS experiments were performed with a LCQDECA (ThermoQuest, San Jose, CA , USA) equipped with a Monitor C18 column (0.2 x 50 mm)at flow rate of 1∼2µl/min. Results: The Mass spectrometric analysis and database search of 400 spots expressed in VH derived from a patients with diabetic retinopathy allowed us to characterize 50 spots. By comparing the 2D-PAGE profiles, many spots were detected in VH, but not in serum. The specific proteins in VH were located at isoelectric points between 5.0 and 9.0, and of molecular weights between 20 kda and 65 kDa. This was a relatively smaller region with higher pI, including more than 300 spots. These were ER81 protein, phosphoglucomutase-related protein, gene pp21, electron tansfer flavprotein-ubiquinone oxidoreductase, dystrophin/utrophin-associated protein, s100 calcium-binding protein and putative HLA class II associated proteins. A protein for regulation of vascular growth was also found, i.e. pigment epithelium derived factor(PEDF). Conclusion: We detected more than 400 spots in humor VH, and iodentified some VH specific proteins as well as major plasma proteins by 2D-PAGE and mass spectrometry. PEDF, a potent antiangiogenic factor, was first characterized in human VH derived from diabetic retinopathy using this technique.Quantitative analyses of the factor are required, and other factors either angiogenic or antiangiogenic must be identified using 2D-gel electrophoreisis.
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