Establishment Of Two-dimensional Electrophoresis System For Comparative Proteomic Analysis Of Caudal Gland

In this study, caudal glands were used as experimental material, firstly, a suitable two-dimensionalelectrophoresis (2-DE) system of caudal gland was eatablished by filtering and optimization theimportant parameters of procedure. Then, the differences of proteins expression in different sexesbetween Cervus elaphus Linnaeus and Cervus Nippon Temminck were studied by means of comparativeproteomic, mass spectrometry and bioinformatics. The experimental methods and results werementioned as follows:Exp.1:7cm pH3-10IPG strips were used to separate caudal gland proteins, and sample preparationmethods, loading amounts, isoelectric focusing conditions and gel consistency were groped andoptimized to establish a proper2-DE system for caudal gland proteomic analysis. The results werementioned as follows:(1) Among the five methods, majorized Trizol extraction was the optimized caudal gland proteinextraction method.(2)200-300μg of proteins loading amount, Isoelectric focusing programⅡand ten percent of gelconsistency was better suited to separate proteins of caudal gland, therefor a proper2-DE system forcaudal gland proteomic analysis was established.(3) The distribution of caudal gland proteins was mainly shown in the area of pH5-8.Exp.2: In this study, the2-DE maps in different sexes of caudal gland beteewn Cervus elaphusLinnaeus Cervus Nippon Temminck were obtained with pH5-817㎝IPG strips based on the established2-DE system. And then the maps were analyzed by PDQest8.0to find the different and specialexpressed protein spots. Finally, mass spectra (MS) and bioinformatics technique were applied toidentify and analysis these proteins spots. The results were as follows:(1) The result of total protein content showed that the protein content had significant differencebetween species (P＜0.05), while no correlation was observed between protein content and sexes (P＞0.05); the content of total protein in caudal gland of Cervus elaphus Linnaeus had significant higherthan that in Cervus Nippon Temminck.(2) PDQest analysis showed that the average protein spots in different samples showed no significantdifference (P＞0.05)； there were five pairs of notable differences in protein distribution and somedifferent expressed protein spots were detected between the four groups of2-DE maps. Typically, theexpression of GST in caudal gland of Cervus elaphus Linnaeus had significant higher than that inCervus Nippon Temminck.(3) Gelsolin, peroxiredoxin2, tissue alpha-L-fucosidase and syntenin were specific proteins in caudalgland of Cervus elaphus Linnaeus, while hsp70and transketolase were specific proteins in caudal glandof Cervus Nippon Temminck.(4) There were70protein spots were successfully identified by MS among the95cutted spots, thesuccess rate was73.68％and they were classified into52species.(5) According to the function,52species of proteins could be divided into five groups and they were 25oxidationresistant and Immune proteins,3transport and storage proteins,5move and supportproteins,10energy and material metabolism proteins, seven protect and defense proteins,9unknown orpredicted proteins, which respectively accounted for48.08%,5.77%,9.61%,19.23%,17.31%.(6) The functional analysis showed that proteins such as peroxiredoxin2, peroxiredoxin6,glutathione S-transferase protein family(GST), hsp70, hsp60, hsp27and albumin play an important rolein antioxidative ability; and proteins such as S100A4, S100A10, S100A11, syntenin, annexinⅠ,annexin Ⅱ and annexinⅣ,alpha enolase,lysoome and ubiquitin play a key role in inflammationregulation, cell proliferation and tumor development.In conclusion, in this paper, sample extraction method and2-DE system for which suitable caudalgland proteomic analysis was successfully established. The comparison of protein content and2-DEmaps from four samples showed that protein spots distribution and abundance had some differences,and some specific proteins were found. This will provide experimental basis and method for identifyingthe species and sexes in Cauda Cervi. At the same time, lots of proteins related to antioxidative ability,and defensive immunity were identified, which will offer theoretical foundation for exploringpharmacological action and revealing detoxication mechanisms of caudal gland.