A Data Normalization Method of Thermal Proteome Profiling

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.