Application of fluorescence difference gel electrophoresis technology in searching for protein biomarkers in chick myopia.
The lens-induced myopia (LIM) in response to concave lens (negative lens) is a well established animal model for studying myopia development. However, the exact visual and neurochemical signaling mechanisms involving myopic eye growth are yet to be elucidated. The feasibility of applying a novel two-dimensional fluorescence difference gel electrophoresis technique for global protein profilings and a search for differential protein expressions in LIM were explored in the present study. Two-dimensional polyacrylamide gel electrophoresis was performed employing a "minimal Lysine labeling" approach and a reverse CyeDye experimental protocol using retinal tissue from chicks. The retinal protein profiles between myopic and control eyes were found to be very similar. More than a thousand protein spots could be detected on a 2D gel. Sixteen and ten protein spots were found to be up-regulated and down-regulated respectively in the myopic eyes according to our preset criteria with the inclusion of an internal pool standard. About 65% of those filtered spots could be successfully identified by peptide mass fingerprinting by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry . Most of the differentially expressed proteins were found to be related to cytoskeletal or oxidative functions. According to the prediction of subcellular locations, most of them (about 84%) were classified as cytoplasmic proteins. The cellular functions for those differentially expressed proteins were reported and their possible involvements in the compensated eye growth were discussed. We have optimized a workable protocol for the study of the differential retinal protein expressions in the LIM using 2D-DIGE approach which was shown to have a number of advantages over the traditional 2D electrophoresis technique.
Lam
Thomas C
TC
Laboratory of Experimental Optometry, Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Hung Hom, Kowloon.
Li
King-Kit
KK
Lo
Samuel C L
SC
Guggenheim
Jeremy A
JA
To
Chi Ho
CH
D000818:Animals; D015415:Biomarkers / Q000737:chemistry*; D002232:Carbocyanines / Q000494:pharmacology*; D002645:Chickens; D003599:Cytoskeleton / Q000378:metabolism; D004586:Electrophoresis / Q000295:instrumentation* / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D007091:Image Processing, Computer-Assisted; D013058:Mass Spectrometry / Q000379:methods; D009216:Myopia / Q000378:metabolism* / Q000473:pathology*; D010100:Oxygen / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods; D012160:Retina / Q000378:metabolism*
D015415:Biomarkers; D002232:Carbocyanines; D005456:Fluorescent Dyes; D010100:Oxygen