Comparative Studies On 2-DE Protein Patterns Of Shrimp Meats

Volume pursuing mass production descends shrimps quality dramatically, thus, it has become hot point of this field no matter home or abroad to find an effective evaluation and grade division method on shrimps quality and to research on quality control and the intrinsic mechanism of shrimps. So far, studies of this field have been generally focused on nutrient analysis, flavor compounds detection and allergen. However, methods which could effectively evaluate the quality of the shrimps and its intrinsic mechanism are still not well established. Featured with high resolution and throughput, two-dimensional gel electrophoresis (2-DE) provides us with the new direction of research. Doing variation analysis of different species and searching mark protein related with the quality of shrimps by using 2-DE technology is of great significance to the quality control, evaluation and grade division of shrimps.This study aimed at establishing the shrimp muscle proteomic methods on sample preparation and 2-DE conditions through comparing and optimizing the relevant technical parameters. Based on it, we have established 2-DE patterns of four shrimp species(Penaeus Vannamei, Metapenaeus Ensis, Macrobrachium Rosenbergii and Macrobrachium Nipponense) and appraised the quality of shrimps by using technologies of differential proteomics. The main results of the experiment were as follows:1. The sample separation of shrimp flesh was established with the relative techniques parameters including sampling part and the lysis solution of 8 M urea,4% CHAPS,40 mM Tris,0.5% Carrier Ampholyte, and 1% TBP, the protein extraction rate reached to (166.68±2.36)μg/mg; Moreover, the optimized purification method was capable of removing impurity and gave more clear background in the 2-DE images by using clean up kit which has the high resolution and good repetition.2. By comparing and optimizing rehydration models, isoelectric focusing (IEF) parameter, SDS-PAGE gel concentration, IPG gel strips and protein loading amount, this study established the 2-DE patterns of shrimps. Satisfactory IEF separation was obtained with reduced horizont-al stripes by using techniques that rehydration model of passive rehydrat-ion for 14 h and IEF method of keeping voltage increasing gradually. The optimized method is that using the nonlinear IPG gel strips 24 cm, pH 4-7 to separate proteins, the loading amount of protein is 220μg while the volume is 450μL and the concentration of SDS-PAGE gel is 12%. With this method, the 2-DE profile was of good resolution and repetition, and the matching rate was more than 75%.3. Separating proteins of four different species of shrimps(Penaeus Vannamei, Metapenaeus Ensis, Macrobrachium Rosenbergii and Macrobrachium Nipponense) with the same protein concentration by 2-DE, the result showed the repeatability (n=3) of triplicate gels for each sample was more than 77% (p<0.05). More than 1300 spots were detected on each silver stained gel. The result indicated that 59 spots showed significant difference among shrimps by using statistical and quantity analysis,46 spots were species-specific.