Rapidly measuring protein thermodynamic stability is vital for understanding the fast protein folding and unfolding processes, involving many human disease-linked protein structures and the fulfilment of their dynamic functions. Heating-induced protein unfolding and conformational transformation studies may generate a variety of protein thermodynamic stability information such as protein melting temperature (T_m) and Gibbs free energy (ΔG). Previous nanoelectrospray ionization (nanoESI) - mass spectrometry (MS) has been interfaced with online heating device to achieve such information, but mostly operating in a slow mode in terms of temperature control. Herein, a new module for digital temperature control (DTC) was constructed and assembled into a nanoESI device, allowing for ultrafast measurement of thermodynamic stability. Typically, DTC can achieve whole-range heating-induced unfolding in 33 s with temperatures ranging from 0 °C to 99 °C but with jump step of 3 °C and deviation less than 1 °C. Notably, thanks to the advantage of ultrafast and precise temperature control, only less than 100 nL protein sample was consumed for each test, saving samples by more than 100 folds compared to previously reported temperature control devices. Besides, with the DTC-nanoESI-MS regime, we successfully achieved solution pH-dependent protein thermodynamics, which serves as first proof-of-concept demonstration for future applications even at a proteome level directly from limited biological samples.

快速测量蛋白质热力学稳定性对于理解蛋白质快速折叠和展开过程至关重要，其中涉及许多与人类疾病相关的蛋白质结构及其动态功能的实现。加热诱导的蛋白质解折叠和构象转变研究可以产生各种蛋白质热力学稳定性信息，例如蛋白质熔化温度（T _m ）和吉布斯自由能（ΔG）。以前的纳电喷雾电离 (nanoESI) -质谱法(MS)已与在线加热装置连接以获取此类信息，但在温度控制方面大多以慢速模式运行。在此，构建了一种新的数字温度控制（DTC）模块并将其组装到 nanoESI 设备中，从而可以超快速测量热力学稳定性。通常，DTC可以在33秒内实现全范围加热诱导展开，温度范围为0°C至99°C，但跳跃步长为3°C，偏差小于1°C。值得注意的是，得益于超快速和精确控温的优势，每次测试仅消耗不到100 nL的蛋白质样品，与之前报道的温控设备相比节省样品100倍以上。此外，利用 DTC-nanoESI-MS 体系，我们成功实现了溶液 pH 依赖性蛋白质热力学，