One typical readout for the thermal stability of a protein is the melting point, or T_m. The T_m is the point at which 50% of the protein (or domain) is unfolded. The melting point by definition describes reversible denaturation processes which are in equilibrium throughout an experiment, typically requiring slow heating rates. These criteria are rarely met for thermal unfolding of proteins, which is typically an irreversible process. On the Tycho NT.6 with its fast heating rate, inflection points therefore do not correspond to the physical melting point of the protein, and are simply called inflection temperatures (T_i) instead. Due to the exceptional reproducibility and repeatability, these T_i serve as very robust parameters for comparing thermal protein stability between Tycho measurements. For researchers to whom T_ms are important, for example to compare to literature values, other equipment is recommended, for example the Prometheus system.

When T_i values measured on Tycho are compared to T_m values measured with other technologies like Prometheus, the T_i typically has a higher temperature value. This is due to the fast heating rates – bluntly speaking, the temperature increases faster than the protein unfolding process can follow. However, it depends on the individual protein and its unfolding kinetics how much difference there is between the two values (see table below). We have seen examples where T_i and T_m were very close together, but also examples where they were around 10 °C apart (with the T_i being higher). Of course both are reproducible and useful, they just aren’t alike.