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Fengjuan Yu, King Kit Li, Thomas Chuen Lam, Chi-ho To; Differentially Protein Expression and Identification in Myopic and Hyperopic Chick Rtina Using Nano-electrospray Ionization Tandem Mass Spectrometry. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5661.
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To find differentially expressed protein candidates in myopic and hyperopic retina of chicks using nano-electrospray ionization tandem mass spectrometry techniques
The right eyes of chicks (3-day old, n=5) wore -10D lenses for 6 days whereas the left eyes wore +10D lenses. The refractive error and ocular biometry were measured using a streak retinoscope and a high frequency ultrasound transducer respectively. Equal amount of retinal protein were labeled with 12C-reagent and 13C-reagent respectively. They were combined together for analysis (SERVA ICPL Kit). Forty bands were cut from the 1-D gel and digested with trypsin (37°) and Glu-C (25°). After extraction, each peptide was dissolved in 0.1% formic acid and subject to nano-electrospray ionization tandem mass spectrometry (ESI-MS/MS) for protein detection. Protein identification and quantification were performed in International Protein Index (IPI) database using WARP-LCTM software.
The spherical equivalent power of right eyes with -10D was significant myopic (Mean ± SEM: -7.50±0.68) and the left eyes with +10D has significant hyperopia (Mean ± SEM: 10.11±0.49). A total of 1063 proteins identified were searched with the IPI chicken database (protein score >75, ≥3 peptides). After data normalization, proteins that have at least 3 identified peptides with ratios and protein ratios exceeded 0.7 to 1.4 were regarded as differentially expressed. Forty-three differentially expressed proteins were identified. Among them phosphoglycerate kinase and L-lactate dehydrogenase A chain, which were related to glycolysis, were up-regulated in myopia.
ESI-MS/MS in combination with 1-D gel provides a powerful tool to identify and detect proteins. A good number of proteins can be identified, which is crucial in understanding the molecular network in myopic eye growth. Although the complete picture of protein changes in myopia is yet to be fully revealed, the results have showed up regulation of enzymes in glycolysis.
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