建立了基于在线二维弱阴离子交换色谱-反相液相色谱(2D-WAX-RPLC)的蛋白质分离系统,并用于不同生长时期鹿茸的比较蛋白质组分析.以5种标准蛋白质的混合物为样品,考察了系统的重现性.通过改变标准蛋白质之间的浓度比,研究了该系统进行蛋白质相对定量的能力.在此基础上,针对4个不同生长时期的鹿茸样品,分别采用5种不同的方法进行蛋白质提取,经2D-WAX-RPLC分离后,收集各生长时期对应蛋白质的峰高最大比超过2的组分.酶解后,采用微柱反相液相色谱-串联质谱(μRPLC-MS/MS)进行肽段的分离鉴定;共从9个差异蛋白质峰中鉴定到了22个差异蛋白质.研究结果表明,利用基于蛋白质水平的在线二维液相色谱分离技术找寻差异蛋白质,具有重现性好、自动化程度高等优点,可用于开展比较蛋白质组学的研究.
A method of on-line two-dimensional weak anion exchange chromatography-reversed-phase liquid chromatography (2D WAX-RPLC) was established,and applied for the comparative proteome study of antlers with four different growing stages.A five-protein-mixture was used as the sample to evaluate the reproducibility of the system.In addition,the capacity of such a system for the relative quantitative analysis was demonstrated by analyzing protein mixtures with different concentration ratios.Furthermore,five different lysis buffers were used to extract proteins from antlers with different growing stages.After the protein separation with 2D WAX-RPLC,the fractions with maximum peak height ratio larger than 2 were collected,digested,and further identified by micro-reversed-phase liquid chromatography-tandem mass spectrometry (μRPLC-MS/MS).In total,22 differential proteins were identified from 9 fractions.Our experimental results demonstrated that 2D WAX-RPLC based protein separation has the advantages of good reproducibility and automation,and might be a complementary method of two-dimensional gel electrophoresis for comparative proteome study.
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