基于荧光标记的大黄鱼氧化肌肉蛋白质双向电泳技术的建立

在确定大黄鱼肌肉蛋白质双向电泳分离的基础上,采用荧光素-5-氨基硫脲(fluorescein-5-thiosemicarbazide,FTSC)对氧化的大黄鱼肌肉蛋白质进行荧光标记和参数优化,进而建立氧化肌肉蛋白质的双向电泳技术体系。结果显示,氧化肌肉蛋白质较佳的双向电泳程序为:蛋白样品采用液氮研磨和裂解液Ⅲ(含8 mol/L尿素、2 mol/L硫脲、4%3-(3-(胆酰胺丙基)二甲氨基)丙磺酸内盐(CHAPS)、65 mmol/L二硫苏糖醇(DTT)和0.2%载体两性电解质)制备;采用FTSC溶液40℃恒温水浴3 h,对氧化蛋白质进行荧光标记,并采用乙醇-乙酸乙酯混合溶液进行洗脱;标记后的蛋白样品采用p H 5~8的固定化p H梯度(IPG)预制胶条上样后,采用等电聚焦程序C(50 V主动水化14 h,500 V、2 h,1 000 V、1.5 h及4 000 V、1 h三段式除盐,6 000 V、0.5 h和10 000 V、1 h两段式升压,10 000 V聚焦80 000 vhr,最后500 V保持10 h)进行第1向分离,再采用12%的聚丙烯酰胺分离胶进行第2向分离;最后所得凝胶经直接荧光扫描和银染后扫描分别得到氧化肌肉蛋白质的荧光图谱和肌肉全蛋白电泳图谱。由该程序获得的双向电泳图谱具有分离度好、蛋白点清晰、分布均匀等优点,为利用双向电泳和蛋白质组学技术分离鉴定氧化蛋白质种类、进而阐明蛋白质氧化机制提供理论依据。

Establishment of Two-Dimensional Gel Electrophoresis Based on Fluorescence Labeling for Oxidized Muscle Proteins of Large Yellow Croaker (Pseudosciaena crocea)

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.