| The quality of fresh pork is a complicated and multipleunit character. Studies on its intrinsic mechanism, evaluation and management are always the hottest research fields of the meat science. So far, such kinds of researches have been generally focused on lean rates and differences in the quality between normal and poor meat. However, methods for effectively evaluating the quality of the fresh pork and its intrinsic mechanism are still not well established as far as we know. Two-dimensional gel electropHoresis (2-DE) and mass spectrometry (MS), provide us with the new thought to study the intrinsic mechanism of fresh pork quality, are the two key technologies of proteomics. With the power of proteomics, the intrinsic mechanism of fresh meat quality and the pHysiological and biochemical basis of genetic regulation were investigated by searching and screening for protein markers of meat quality traits. Thus proteomics offers a related foundation for finding quality genes through cDNA.Therefore, this study was conducted to evaluate the quality of fresh pork of two different species (Jinhua pigs in the original area of Zhejiang, China, termed as JH, and hybrid pigs of Duroc×Landrace×Yorkshire, termed as DLY) with different feeding periods by using technologies of differential proteomics. We have established the pork muscle proteomic methods on sample preparation and 2-DE conditions through comparing and optimizing the relevant technical parameters. The main results of the present study were as follows.1. The sample separation of fresh pork musle was established with the relative techniques parameters including liquid nitrogen grinding and the lysis solution of 8M urea, 2M thiourea and 4% CHAPS, the protein extraction rate reached to 52.96±0.34μg/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. The 2-DE methods of fresh pork muscle were established with relative techniques optimization including rehydration models, isoelectric focusing (IEF) parameter and staining protocol. Satisfactory IEF separation was abtained with reduced horizontal stripes by using techniques that rehydration model of active rehydration for 6h and passive rehydration for 6h and IEF method of keeping voltage increasing gradually, while more low abundance proteins were detected by modified sliver staining which is suitable for gel analysis.3. Proteins form JH and DLY muscle with same protein concentration were separated by 2-DE, the repeatability (n=3) of triplicate gels for each sample was 75% (p<0.05). 26 spots showed a significance difference between the fresh pork of JH and DLY by using statistical and quantity analysis. More than 580 spots were detected in each jinhua pig postmortem storage groups, 9 spots showed a significant (p<0.05) difference during 72h postmortem storage. Based on average value for each spot, two spots at jinhua pigs group weren't detected at one or two-points, including one at Oh group, one at oh, 24h group. One spot showed the highest protein expression at 48h group. 4 spots were consistently reduced during ageing, while 2 spots were consistently increased during ageing. 8 proteins were separated from JH muscle of different weight, which showed significant difference (p<0.05). |
