一种热蛋白质组学分析数据的归一化方法

A Data Normalization Method of Thermal Proteome Profiling

建立了一种小分子靶标蛋白的鉴定方法,即引入索引保留时间(Indexed retention time,iRT)肽段作为非标记热蛋白质组学(Thermal proteome profiling,TPP)分析定量归一化内标,实现对小分子靶标的准确挖掘.将iRT肽段加入到表征良好的模型药物甲氨蝶呤(Methotrexate,MTX)处理的293T细胞提取液中,在不同温度加热处理时它们稳定存在,因此可作为定量归一化的标准.实验结果表明,以iRT作为定量校正,不仅对总蛋白含量有良好的校正效果,同时对MTX的靶标蛋白二氢叶酸还原酶(Dihydrofolate reductase,DHFR)热熔解拟合曲线也有一定改善.该方法简单方便、经济高效,具有较强实用性和推广性,为非标记TPP技术的实施及定量准确性提供了重要的实验依据.

We developed a simple,cost-effective method with wide application for the accurate identification of small molecule target proteins,in which indexed retention time(iRT)peptides were adopted to be an internal standard for quantification normalization in label-free quantitative thermal proteome profiling(TPP)analysis.The iRT standard peptides were supplemented into the lysates of 293T cells treated by a well-characterized model drug methotrexate(MTX).These peptides were quite stable during heating under different temperatures and it is reasonable to use them for quantification normalization.The results showed that iRT peptides used as an internal standard provided a good normalization effect on the global proteomes.Meanwhile,the melting curve of the target protein dihydrofolate reductase(DHFR)of MTX has been improved.This method provides an important experimental basis for the implementation and quantitative accuracy of label-free TPP techniques.