基于iTRAQ技术的水牛卵泡液蛋白质样品预处理的优化(英文)  

Optimization of protein sample preparation for buffalo follicular fluid analyzed with iTRAQ technique

作　　者：胡炜[1,2] 周洁[1,2] 付强[1,2] 

机构地区：[1]亚热带农业生物资源保护与利用国家重点实验室,南宁530005 [2]广西大学,南宁530005

出　　处：《南方农业学报》2014年第11期2046-2051,共6页Journal of Southern Agriculture

基　　金：Scientific Research Fund of Guangxi Education Department(YB2014004);The Open Topic from Guangxi Key Laboratory of Buffalo Genetics Reproduction and Breeding(SNKF-2012-05,SNKF-2013-02)

摘　　要：【目的】优化水牛卵泡液蛋白质差异表达样品制备程序,为相对和绝对定量同位素标签(iTRAQ)技术在蛋白质差异表达分析中的应用奠定基础。【方法】在蛋白质样品溶液酶解过程中,选择两种不同沉淀剂沉淀蛋白质,分别比较蛋白质酶解、iTRAQ标记反应和多肽色谱分离的效果;在纳升液相色谱(nano-LC)分离样品前用快速分离小柱进行处理,鉴定色谱分离效果,以确定蛋白质样品预处理的优化程序。【结果】相对于丙酮,使用含0.1%乙酸的丙酮—乙醇混合液(1∶1)作为沉淀剂可获得较好的蛋白质沉淀效果,蛋白沉淀为白色,蛋白质浓度为5.76μg/μL。相对于蛋白质溶液在还原烷基化后直接使用胰蛋白酶酶解,在酶解前沉淀蛋白质溶液可使蛋白样品的酶解效率更高,获得的肽段峰更丰富、峰值更高;可使多肽和iTRAQ试剂间产生有效的标记反应,母离子具有较高的碎裂效率,获得的二级碎片峰更丰富,iTRAQ试剂的特征报告离子116 m/z较117 m/z的谱图强,辨识度高;获得的多肽nano-LC分离色谱峰更丰富,峰值和分辨率更高。相对于直接进行LC分离,在nano-LC分离前用快速分离小柱对多肽溶液进行杂质处理,也可获得丰富的多肽nano-LC色谱峰,且峰值和分辨率较高。【结论】在iTRAQ试剂标记、蛋白质液相分离和差异分析前,使用适宜的沉淀剂处理蛋白质溶液,并以快速分离小柱除去多肽混合液小分子杂质,可使质谱获得更准确的鉴定结果。[ Objective ]The optimization of protein sample preparation extracted from buffalo follicular fluid was conducted to study differential expression protein by using isobaric tags for relative and absolute quantification （iTRAQ ） technique in order to lay the foumdation for iTRAQ application on protein differential expression analysis. [ Method ]Two different precipitants were used for precipitating protein in the process of protein solution enzymolysis to compare their effects on protein enzymolysis, iTRAQ labeling reaction and chromatographic separation of peptides. The peptides solution was treated with speed column before separated by nano liquid chromatography （nano-LC） to observe its chromatograph separation effect in order to confirm the optimal procedure of protein sample pretreatment. [Result]As compared to use 100% acetone as the precipitant, the protein solution precipitated with 1：1 volume ratio of acetone and ethanol mixture adding 0.1% acetic acid could obtain ideal precipitation effects being white precipitation and 5.76 μg/μL of protein content. Compared with no precipitation of protein solution after reduction and alkylation in the process of enzymolysis, the higher enzymolysis efficiency, and much peptide peaks with higher intensity could be found in separation chromatogram when the protein solution was precipitated with acetone and ethanol mixture before trypsin enzymolysis. As a result, the labeling reaction between iTRAQ reagent and peptide was very effective to get higher fragmenting efficiency of precursor ion and much fragmental peaks, and the iTRAQ characteristic reporter ion of 116 m/z showed better spectrogram peak intensity and higher resolution than that of 117 m/z. Furthermore, the liquid chromatogram of peptides from precipitated protein solution presented much peaks with higher intensity and distinguishability, h found that much chromatographic peaks, higher peak intensity and distinguishability were presented in the nano-LC chromatogram of peptides solution by removing s

关 键 词：水牛卵泡液 蛋白质样品制备 沉淀剂 酶解 iTRAQ标记 优化 

分 类 号：Q51[生物学—生物化学]