Proteome Analysis Of Hemolymph Plasma Proteins Of Chinese Shrimp, Fenneropenaeus Chinensis

With the completely sequencing of human genome,a new era -post-genome era is coming.The key task of life science transferred from large scale sequencing of genome to the functional characterization of genes.Proteome has become one of the hot fields in life sciences.Proteomics represents a novel methodological approach to investigate the expression of all proteins of a cell,body fluid or organism,to analyze their structure, function,interaction and modification.Proteomics has progressed enormously in recent years and been a favorite research means for post-genomics.Plasma,as a major component of hemolymph,plays important physiological functions.Plasma proteins would take place some quality and quantity changes when suffering from pathological damages of shrimp.Detecting these characteristic changes is critical for disease diagnosis and therapeutic monitoring.At the same time, plasma is a complicated and dynamic changed system,consists of different kind and abundant proteins,which bring out many difficulties for the plasma research.The development of proteomic technologies provide favorable platforms to understand plasma proteins in healthy and sick states.First,key techniques in the proteomics study such as 2-DE,MS,bioinformatics, and other related techniques have been reviewed.Applications of genomics and proteomics in shrimp species are summarized.second,we have developed an efficient approach for sample preparation and attempt to study plasma protein electrophoretic patterns of Chinese shrimp Fenneropenaeus chinensis;we systematically studied tryptic peptides elution condition and the characteristics of identified peptides in reverse phase liquid chromatography and electrospray ion trap tandem mass spectrometry(LC/MS/MS) analysis;After in-gel digestion,A total of twenty proteins were identified by ESI-MS/MS.Database search and characterization of the proteins identified suggest that these proteins might be mainly involved in such important processes;The identification method for protein mixture is reported,in which several proteins were varified by HPLC-ESI/MS/MS.The whole complex proteins were directly digested in solution,instead of the routine 2-dimensional gel electrophoresis.The obtained data was then searched against NCBI nr database,and a positive result was therefore obtained.SDS-PAGE pre-fractionation was added to achieve a more comprehensive protein profile of plasma proteins.A total of 66 non-redundant proteins were identified.(1)We have developed an efficient approach for sample preparation and attempt to study plasma protein electrophoretic patterns of Chinese shrimp Fenneropenaeus chinensis.The proteins extracted from hemolymph separated by two-dimensional electrophoresis,and the conditions of isoelectric focusing and SDS-PAGE were discussed and optimized.The results showed that higher protein extraction rate and better reproducibility were acquired in the present experiment.Using our sample preparation,2-DE gel images of plasma showed an excellent reproducibility.(2)We systematically studied tryptic peptides elution condition and the characteristics of identified peptides in reverse phase liquid chromatography and electrospray ion trap tandem mass spectrometry(LC/MS/MS)analysis.Following protein digestion with trypsin,the peptide mixture was analyzed by on-line LC/MS/MS.AgI had been used to develop the proper ACN elution gradient for tryptic peptides and Bovine serum albumin(BSA)was used to prove the gradient and the sensitivity of LC/MS/MS.In addition,the characteristics of identified peptides were analyzed.In our experiments the sensitivity of LC/MSIMS was 50 fmol for BSA.(3)After 2DE separation,plasma proteins of Chinese shrimp Fenneropenaeus chinensis were identified by ESI-MS/MS.Result suggest that these proteins may function as different roles in shrimp physiological processes though diverse routes they participate in.The present work would deliver important data for better insights into mechanisms of physiological processes,and is of significance in finding out and mining of biological markers.(4)The identification method for protein mixture is reported,in which several proteins were varified by HPLC-ESI/MS/MS after the whole complex proteins were directly digested in solution,instead of the routine 2-dimensional gel electrophoresis. The obtained data was then searched against NCBInr database,and a positive result was therefore obtained.SDS-PAGE pre-fractionation was added to achieve a more comprehensive protein profile of plasma protein.A total of 66 non-redundant proteins were identified.Proteins like to be associated with Transcription,Structural and Cytoskeletal,Signal transduction,Protein synthesis and processing,Protein folding,sorting and degradation,Immune function,Growing and development, Energy metabolism,Cell cycle,Carbohydrate metabolism,Calcium homeostasis, Antioxidant and Amino acid and lipid metabolism.Third,hemocyanins(Hcs)was studied.Hcs which responsible for oxygen transport,are a kind of extracellular giant proteins found in the hemolymph of both mollusks and arthropods.Hemocyanin from hemolymph of Fenneropenaeus chinensis was purified by Sephacryl S-100 gel-filtration.It was found that the purified Hc had two bands in SDS-PAGE(sodium dodecyl sulfate polyacrylemide gel electrophoresis)and it was composed of three subunits analyzed by 2DE. Furthermore,proteins were identified by LC-ESI-MS against the NCBI database which confirmed that these three subunits were identical to partial fragment of hemocyanin from Litopenaeus vannanmei.It can be concluded that the protein purified in this study does be hemocyanins.