Scaled-Down Thermal Profiling and Coaggregation Analysis of the Proteome for Drug Target and Protein Interaction Analysis

Lu, Xue; Liao, Bin; Sun, Siyuan; Mao, Yiheng; Wu, Qiong; Tian, Ruijun; Tan, Chris Soon Heng

2023-09-01

10.1021/acs.analchem.3c01941

ANALYTICAL CHEMISTRY   Impact Factor & Quartile

0003-2700

1520-6882

Thermal proteome profiling (TPP), an experimental technique combining the cellular thermal shift assay (CETSA) with quantitative protein mass spectrometry (MS), identifies interactions of drugs and chemicals with endogenous proteins. Thermal proximity coaggregation (TPCA) profiling extended TPP to study the intracellular dynamics of protein complexes. In TPP and TPCA, samples are subjected to multiple denaturing temperatures, each requiring over 100 mu g of proteins, which restricts their applications for rare cells and precious clinical samples. We developed a workflow termed STASIS (scaled-down thermal profiling and coaggregation analysis with SISPROT) that scales down the required protein to as low as 1 mu g per temperature. This is achieved by heating and centrifugation using the same PCR tube, processing samples with the SISPROT technology (simple and integrated spintip-based proteomics technology), and tip-based manual fractionation of TMT-labeled peptides. We evaluate the STASIS workflow with starting protein quantities of 10, 5, and 1 mu g per temperature prior to heating, identifying between 4000 and 5000 proteins with 6 h of acquisition time. Importantly, we observed a high correlation in the T-m of proteins with minimal difference in TPCA performance for predicting protein complexes. Moreover, STASIS could identify the targets of methotrexate and panobinostat with high precision with 1 mu g of proteins per temperature. In conclusion, STASIS is a robust cost-effective technique for target deconvolution and extended TPCA to rare primary cells and precious clinical samples for the analysis of protein complexes.

SCI

English

NI Journal Papers

First ; Corresponding

National Key Research and Development Program of China[2021YFA1302603] ; Shenzhen Innovation of Science and Technology Commission Grant[JCY20200109140814408] ; National Natural Science Foundation of China["22074060","22150610470"]

Chemistry

Chemistry, Analytical

WOS:001061207400001

AMER CHEMICAL SOC

CHEMISTRY

Web of Science

Southern Univ Sci & Technol, Dept Chem, Shenzhen 518055, Peoples R China

Lu, Xue,Liao, Bin,Sun, Siyuan,et al. Scaled-Down Thermal Profiling and Coaggregation Analysis of the Proteome for Drug Target and Protein Interaction Analysis[J]. ANALYTICAL CHEMISTRY,2023.

Lu, Xue.,Liao, Bin.,Sun, Siyuan.,Mao, Yiheng.,Wu, Qiong.,...&Tan, Chris Soon Heng.(2023).Scaled-Down Thermal Profiling and Coaggregation Analysis of the Proteome for Drug Target and Protein Interaction Analysis.ANALYTICAL CHEMISTRY.

Lu, Xue,et al."Scaled-Down Thermal Profiling and Coaggregation Analysis of the Proteome for Drug Target and Protein Interaction Analysis".ANALYTICAL CHEMISTRY (2023).