| Chinese shrimp (Fenneropenaeus chinensis) is one of the main cultured shrimps in China. With its rich nutrition, it is favored by many consumers. However, it is easy to decay and lose freshness like many other aquatic products. So many kinds of methods of evaluating the quality of the aquatic products have been developed, such as sensory evaluation, physical evaluation, chemical evaluation, and microbial evaluation, et al. However, little information is available on the fressness assessment on aquatic products based on muscle protein.In recent years, proteomics, which is composed by two-dimensional electrophoresis (2-DE), mass spectrometry (MS) and bioinformatics, has got a fast development and provided us with new thoughts to study the intrinsic mechanism of quality changes based on muscle protein in aquatic products. To develop a new freshness assessing method of Chinese shrimp, we can screen freshness related protein by proteomics, and do some further studies on the biochemical mechanisms of quality changing and freshness losing by employing database of protein.A two-dimensional electrophoresis technologic platform on muscle protein of Chinese shrimp was established in this study by comparing and optimizing relevant technical parameters effecting it. Low-salt-soluble proteins and whole muscle proteins of Chinese shrimp were extracted during storage and preservation respectively. Proteomic studies on the two kinds of proteins were earried out. The main results of the present study were as follows.1. The techniques of two-dimensional electrophoresis (2-DE) of muscle protein in Chinese shrimp were constructed. The formulation of lysis buffer was made up of8M urea,2M thiourea,4%CHAPS,65mM DTT,0.67%Bio-Lyte. Muscle protein sample of120ug in300μL was loaded in17cm (pH4-7) IPG gel strip, which was connected by salt bridge. The optimum time of IEF was80000v*h and equilibration was13.5min. During SDS-PAGE,12.5%SDS-PAGE gels were prepared, and the gels were dyed by the silver stained. Then the maps of gels were obtained and analyzed by the PDQuest software. The2-DE method can provide a high resolution of2-DE maps for Chinese shrimp muscle protein.2. Significant changes were found in the low-salt-soluble protein in Chinese shrimp during storage by preliminary SDS-PAGE analyse. Thus the2-DE method was employed to study the proteomic changes of the proteins. Eight differently expressed protein spots were found in the2-DE maps. Peptide mass fingerprintings were obtained by MALDI-TOF-MS. One protein was identified to be NKG2D, which was related to immune system of organism and had changed significantly from largest to smallest at the storage time of8days. It was considered to be the freshness related protein of Chinese shrimp.3. The former study has found that chitosan coating had a significant effect on preservation for Chinese shrimp, so the2-DE was used to analyze the difference of muscle proteins in Chinese shrimp during storage treated by chitosan coating or not. The differential proteins were chosen from three ways of comparisons, comparisons among shrimps treated by chitosan coating at different storage times, comparisons among shrimps not treated by chitosan coating at different storage times, comparisons among shrimps treated by chitosan coating or not at the same storage time.8differential proteins were statistically significant (P<0.05) and peptide mass fingerprintings were obtained by MALDI-TOF-MS. At last, one protein was identified, it was ATP-PRT (ATP phosphoribosyltransferase) which was related to histidine and ATP metabolism. It was considered to be the freshness related protein of Chinese shrimp. |
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