Wang Qiqi, Chen Qiufen, Lin Yue, He Dan, Ji Hongchao, Tan Chris Soon Heng
Department of Chemistry and Research Center for Chemical Biology and Omics Analysis, College of Science, Southern University of Science and Technology, Shenzhen, Guangdong 518055, P.R. China.
Shenzhen Key Laboratory of Functional Proteomics, Guangming Advanced Research Institute, Southern University of Science and Technology, Shenzhen 518055, China.
Anal Chem. 2024 Dec 10;96(49):19695-19705. doi: 10.1021/acs.analchem.4c04837. Epub 2024 Dec 1.
Target deconvolution is essential for elucidating the molecular mechanisms, therapeutic efficacy, and off-target toxicity of small-molecule drugs. Thermal proteome profiling (TPP) is a robust and popular method for identifying drug-protein interactions. Nevertheless, classical implementation of TPP using isobaric labeling of peptides is tedious, time-consuming, and costly. This prompts the adoption of a label-free approach with data-independent acquisition (DIA), but with substantial compromise in protein coverage and precision. To address these shortcomings, we improvised a spike-in proteome strategy for DIA with TPP to counteract the reduction in protein quantity following sample heating. Protein coverage, data completeness, and quantification precision are significantly improved as result. Additionally, a calibration algorithm was developed to correct for spike-in effects on fold changes. The integration of DIA-TPP with the matrix-augmented pooling strategy (MAPS) to increase experiment throughput demonstrates performance comparable to that of existing TMT-TPP-MAPS. With this spike-in proteome strategy, we also successfully identified the thermal stabilization of CA13 by dorzolamide hydrochloride as well as GSTZ1 and tyrosyl-DNA phosphodiesterase 1 of opicapone that eluded detection without spike-in proteome.
