关键词: 双向电泳, 大黄鱼, 氧化肌肉蛋白质, 荧光素-5-氨基硫脲, 荧光标记

Abstract: The purpose of this study was to optimize the processing parameter for fluorescence labeling of oxidized muscle proteins from large yellow croaker (Pseudosciaena crocea) with luorescein-5-thiosemicarbazide (FTSC), and further to establish a two-dimensional gel electrophoresis (2-DE) system for oxidized muscle proteins. The results showed that the optimized electrophoresis process was as follow. The protein sample was prepared by liquid nitrogen milling and using a lysis buffer containing 8 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 65 mmol/L DTT and 0.2% carrier ampholyte, and then the oxidized protein was labeled with FTSC (20 mmol/L in DMSO) for 3 h at 40 ℃ in the dark, and washed five times with ethanol/ethyl acetate (1:1). The sample was loaded onto immobilized pH gradient (IPG) gel strip (pH 5–8), and separated by isoelectric focusing (active rehydration for 14 h at 50 V; desalting for 2 h at 500 V, 1.5 h at 1 000 V and 1 h at 4 000 V; voltage for 0.5 h at 6 000 V at first and then for 1 h at 10 000 V; focusing for 80 000 vhr at 10 000 V; finally balancing for 10 h at 500 V). After isoelectric focusing, the IPG strip was transferred and the proteins were separated by 12% SDS-PAGE. Finally, 2-DE maps for the oxidized and whole muscle proteins with high resolution and even distribution were obtained by direct scanning using a fluorescence image scanner and scanning after silver staining. This study would provide a foundation for the separation and identification of oxidized muscle proteins, and further for the clarification of protein oxidation mechanism by two-dimensional gel electrophoresis and proteomics technology.

Key words: two-dimensional electrophoresis, large yellow croaker (Pseudosciaena crocea), oxidized muscle proteins; fluorescein-5-thiosemicarbazide (FTSC), fluorescence labeling