样品前处理对牛精子全蛋白质组鉴定的影响  

Effect of Sample Preparation Approach on Bovine Sperm Global Proteomics

作　　者：海超 张洁 刘春丽 李欣 张媛 白春玲 李光鹏 赵跃芳 HAI Chao;ZHANG Jie;LIU Chun-li;LI Xin;ZHANG Yuan;BAI Chun-ling;LI Guang-peng;ZHAO Yue-fang(State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock,Inner Mongolia University,Hohhot 010070,China)

机构地区：[1]内蒙古大学省部共建草原家畜生殖调控与繁育国家重点实验室

出　　处：《内蒙古大学学报（自然科学版）》2019年第4期337-345,共9页Journal of Inner Mongolia University：Natural Science Edition

基　　金：国家自然科学基金项目(31560333);内蒙古自治区自然科学基金(2017MS0318);内蒙古科技厅重大专项省部共建草原家畜生殖调控与繁育国家重点实验室自主课题[ZDZX2018065(SKLIT-201812)]

摘　　要：本文主要分析样品前处理对牛精子全蛋白质组鉴定的影响,文中对5种不同的蛋白质提取液和3种不同的酶解方法进行了系统地分析比较.5种蛋白质提取液:溶液BS(4%SDS,0.1mol/L Tris-HCl,0.1mol/L DTT);溶液BU(8mol/L Urea,0.1mol/L Tris-HCl);溶液BT(7mol/L Urea,2mol/L Thiourea,0.1mol/L Tris-HCl,0.4%Chaps);溶液BR(购自Thermo,货号89901);溶液BD(2%SDC,50mmol/L NH4HCO3,25mmol/L NaCl).3种酶解方法:溶液内酶解、胶内酶解以及膜上酶解(FASP).实验结果显示:牛精子蛋白质提取能力BS最强,BU最弱;BT、BR以及BD相当.BS-FASP不适用于牛精子蛋白质组学研究.胶内酶解鉴定到的蛋白质均多于FASP法,但是胶内酶解操作过程复杂繁琐,耗时长.基于BD的溶液内酶解法鉴定到的蛋白质数量最多,膜蛋白提取鉴定能力较强,且操作时间最短,是较为理想的方法.The effect of different sample preparation approaches on bovine sperm global proteomics was evaluated in this study.Five different protein extracts and three enzymatic digestion approaches were studied and compared systematically.The five protein extracts were buffer BS(4%SDS,0.1 mol/L Tris-HCl,0.1 mol/L DTT),buffer BU(8 mol/L Urea,0.1 mol/L Tris-HCl),buffer BT(7 mol/L Urea,2 mol/L Thiourea,0.1 mol/L Tris-HCl,0.4% Chaps),buffer BR(Thermo,#89901)and buffer BD(2% SDC,50 mmol/L NH4 HCO3,25 mmol/L NaCl).The three enzymatic digestion approaches were digestion in solution,in gel and using FASP.The results showed that,BS had the strongest bovine sperm proteins extraction capability while BU had the weakest.BS-FASP was not suitable for bovine sperm proteomic studies.The proteins identified were always more by ingel digestion than FASP regardless of different protein extracts,but the sample processing of in-gel digestion was complicated and time-consuming.In conclusion,BD-SOLUTION combined with in-gel digestion was an ideal approach for bovine sperm global proteomic study,since its identified proteins were the most,membrane protein extraction efficiency was the high and the time of sample preparation was short.

关 键 词：牛精子 蛋白质组学 样品前处理 蛋白质提取液 酶解方法 

分 类 号：S823.3[农业科学—畜牧学] Q954.43[农业科学—畜牧兽医]