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Application of Fluorescence Difference Gel Electrophoresis Technology in Searching for Protein Biomarkers in Chick Myopia

Supporting Info

Thomas C. Lam,^† King-Kit Li,^† Samuel C. L. Lo,*^‡ Jeremy A. Guggenheim,^§ and Chi Ho To*^†

Laboratory of Experimental Optometry, Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Hung Hom, Kowloon, HKSAR, The Proteomic Task Force, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, HKSAR and The State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Shenzhen, China, and Department of Optometry & Vision Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, U.K.

J. Proteome Res., 2007, 6 (11), pp 4135–4149

DOI: 10.1021/pr0701097

Publication Date (Web): October 9, 2007

†

School of Optometry, The Hong Kong Polytechnic University.

To whom correspondence should be addressed. (Prof. Chi-Ho To) E-mail: sochto@inet.polyu.edu.hk. Tel: (852) 2766 6102. Fax: (852) 2764 6051. Address: School of Optometry, the Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China; (Prof. Samuel Chun-Lap Lo) E-mail: bcsamlo@inet.polyu.edu.hk. Tel: (852) 2766 6686. Fax: (852) 2364 9932. Address: Department of Applied Biology and Chemical Technology, the Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China. (Both authors contributed equally to this work.)

‡

Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University.

Cardiff University.

Abstract

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.

Keywords: Two-dimensional gel electrophoresis • chick • myopia • proteomics • DIGE • retina • mass spectrometry

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