Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China.
Anal Chem. 2023 Sep 19;95(37):13844-13854. doi: 10.1021/acs.analchem.3c01941. Epub 2023 Sep 1.
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 μg 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 μg 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 μg 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 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 μg 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.
热蛋白质组谱分析（TPP）是一种将细胞热转移分析（CETSA）与定量蛋白质质谱（MS）相结合的实验技术，用于鉴定药物和化学物质与内源性蛋白质的相互作用。热接近共聚集（TPCA）分析将 TPP 扩展到研究蛋白质复合物的细胞内动力学。在 TPP 和 TPCA 中，样品要经过多个变性温度处理，每个温度需要超过 100μg 的蛋白质，这限制了它们在稀有细胞和珍贵临床样本中的应用。我们开发了一种称为 STASIS（基于 SISPROT 的缩小热分析和共聚集分析）的工作流程，该流程将所需的蛋白质减少到每个温度低至 1μg。这是通过使用相同的 PCR 管进行加热和离心，使用 SISPROT 技术（简单且集成的基于自旋点的蛋白质组学技术）处理样品，以及基于 tip 的 TMT 标记肽的手动分段来实现的。我们使用每个温度起始蛋白质量为 10、5 和 1μg 来评估 STASIS 工作流程，在 6 小时的采集时间内鉴定出 4000 到 5000 种蛋白质。重要的是，我们观察到在预测蛋白质复合物方面，STASIS 表现出高度相关性，且 TPCA 性能的差异极小。此外，STASIS 可以在每个温度仅使用 1μg 蛋白质的情况下，高精度地识别甲氨蝶呤和帕比司他的靶标。总之，STASIS 是一种强大的、具有成本效益的技术，可用于目标分解以及扩展到稀有原代细胞和珍贵临床样本，以分析蛋白质复合物。
