Experimental strategies to improve drug-target identification in mass spectrometry-based thermal stability assays

Mass spectrometry (MS)-based thermal stability assays have recently emerged as one of the most promising solutions for the identification of protein-ligand interactions. Here, we have investigated eight combinations of several recently introduced MS-based advancements, including the Phase-Constrained Spectral Deconvolution Method, Field Asymmetric Ion Mobility Spectrometry, and the implementation of a carrier sample as improved MS-based acquisition approaches for thermal stability assays (iMAATSA). We used intact Jurkat cells treated with a commercially available MEK inhibitor, followed by heat treatment, to prepare a set of unfractionated isobarically-labeled proof-of-concept samples to compare the performance of eight different iMAATSAs. Finally, the best-performing iMAATSA was compared to a conventional approach and evaluated in a fractionation experiment. Improvements of up to 82% and 86% were demonstrated in protein identifications and high-quality melting curves, respectively, over the conventional approach in the proof-of-concept study, while an approximately 12% improvement in melting curve comparisons was achieved in the fractionation experiment.

基于质谱（Mass Spectrometry，MS）的热稳定性测定法，近来已成为蛋白质-配体相互作用鉴定领域极具前景的解决方案之一。本研究针对新近提出的数项基于质谱的热稳定性测定采集改进方案（improved MS-based acquisition approaches for thermal stability assays，iMAATSA）构建了八种组合形式展开探究，其中涵盖相位约束光谱反卷积法（Phase-Constrained Spectral Deconvolution Method）、场不对称离子迁移谱（Field Asymmetric Ion Mobility Spectrometry），以及载体样品的引入策略。我们采用经市售MEK抑制剂处理的完整Jurkat细胞，辅以热处理流程，制备了一组未分级等压标记的概念验证样本，用于对比八种不同iMAATSA组合的性能表现。最后，本研究将表现最优的iMAATSA组合与传统方法进行对比，并通过分级分离实验对其开展评估。在本次概念验证研究中，相较于传统方法，该方案在蛋白质鉴定与高质量解链曲线获取方面分别实现了最高82%与86%的性能提升；而在分级分离实验中，其在解链曲线对比分析方面亦取得了约12%的性能改进。