Measuring protein unfolding thermodynamic stability in one minute with digital temperature control-equipped nanoESI-mass spectrometry

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.