Shotgun方法在正常人泪液蛋白质组学分析中的应用

Application of Shotgun strategy on tear proteomic analysis in normal people

目的探讨Shotgun方法用于分析泪液蛋白质组,完善正常人泪液蛋白质表达谱。方法收集正常8人(8眼)泪液混合后用于分析,所有人泪液标本均根据自愿的原则,采用毛细管从每眼收集15μL泪液,用Shotgun方法检测泪液蛋白质:分别行溶液内酶解、反向高效液相色谱分离、电喷雾离子化质谱,所得数据搜索IPI人类蛋白库,并按照IPI号对蛋白进行GOA功能分类。结果正常人泪液中共有352个蛋白质,高可信度蛋白质为73个,包括已知的高丰度蛋白质和泪液特异性蛋白质。参与生理过程和细胞过程的蛋白质占到泪液蛋白质总量的83.5%。鉴定出的蛋白质主要分布于细胞质中,已知最多的蛋白质为具有结合功能的泪液蛋白质。结论 Shotgun方法可有效分离和分析泪液蛋白质,该研究完善了成年人正常泪液蛋白质表达谱。

Objective To investigate the Shotgun strategy for the analysis of tear proteome,complete protein expression profile of normal human tear.Methods Tears were obtained from 8 normal controls（8 eyes）and were mixed for analysis.All human tear samples were obtained with the individual＇s consent.About 15 μL tears was collected by microcapillary tubes from each eye.Shotgun strategy was used for tear protein analysis.Trypsin digestion in-solution,separation of peptide mixture by reverse-phase high-pressure liquid chromatograph followed by electrosprayionization mass spectrometry identification.The acquired MS/MS spectra were searched against a protein database for IPI human proteins.Functional classifications were performed with GOA according to the accession number of proteins in IPI.Results About 352 proteins were existed in the normal controls.The number of high confidence protein was 73,including known high abundance proteins and tear-specific proteins.Proteins involved in physiological process and cellular process were accounted for 83.5% of the total tear proteins.The identified proteins were mainly distributed in the cytoplasm.The most known proteins were having the binding function tear proteins.Conclusion Shotgun strategy can separate and analysis tear proteins effectively,which improved the tear protein expression profile of normal adults.