Establishment of two-dimensional gel electrophoresis technique for Fenneropenaeus chinensis plasma and identification of major high-abundance proteins by MALDI-TOF/TOF MS

Chinese shrimp Fenneropenaeus chinensis is one of the most economically valuable and widely cultured species in China.The hemolymph is the source of its immunity to disease and environmental factors,which invariably involves protein components.To better understand the physiology of F.chinensis and the major protein components expressed during its life cycle,two proteomics approaches,two-dimensional gel electrophoresis(2-DE)in conjunction with MALDI-TOF/TOF MS,were employed to analyze its hemolymph proteins.The proteins of F.chinensis plasma were separated using immobilized pH gradient 2-DE after desalting.By optimizing loading amount,and separation scope of IPG strip,the proteins were successfully extracted from Chinese shrimp plasma and were separated by 2-DE.After silver staining or colloidal brilliant blue staining,PDQuest image analysis software was applied to analyze the 2-DE images and a total of 11 proteins highly expressed in shrimp plasma were identified by MALDI-TOF/TOF MS,which are all hemocyanins.The results revealed that when a 150 μg sample,with 18 cm IPG(pH 4-7),after silver staining,2-DE analysis patterns were obtained；and when a 1 000 μg sample,with 18 cm IPG(pH 4-7),after colloidal brilliant blue staining,2-DE prepared patterns were obtained.We have successfully established a 2-DE technique for shrimp plasma proteome,which can lay the foundation for the further study of plasma proteomics of shrimp and other crustaceans.