微尺度热泳(MST),和密切相关的温度相关强度变化(TRIC),是生物物理学领域最近开发的量化生物分子相互作用的测量技术的同义词,使用(基于毛细管的)NanoTemper整体和(基于多孔板的)Dianthus仪器。尽管该技术由于其样品消耗低而在科学界广泛使用,易用性,和无处不在的适用性,MST/TRIC尚未获得生物物理学家对其他生物物理技术的明确认可,例如等温滴定量热法(ITC)或表面等离子体共振(SPR)。这可能归因于几个事实,例如,各种(未完全理解的)效应对信号有贡献,该技术仅授权给单个仪器开发人员,NanoTemper技术,它的可靠性和可重复性从未被独立和系统地测试过。因此,ARBRE-MOBIEU的一个工作组已经建立了MST/TRIC的基准研究,以评估该技术作为表征生物分子相互作用的方法。在这里,我们介绍了这项研究的结果,该研究涉及欧洲的32个科学小组和美国的两个小组,在40台整体仪器上进行实验,采用标准操作程序和集中准备的样品。蛋白质-小分子相互作用,新开发的蛋白质-蛋白质相互作用系统和纯染料被用作测试系统。我们表征了仪器性能并评估了仪器性能,再现性,不同分析工具的效果,实验者在数据分析过程中的影响,因此这种方法的整体可靠性。
Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.