Advanced thermal analysis methods, such as temperature modulated DSC (differential scanning calorimetry) and quasi-isothermal
TMDSC were used to analyze the protein–metallic ion interactions in silk fibroin proteins. The precise heat capacities were
measured and theoretically predicted in this study. To remove bound water and simplify the system, a thermal cycling treatment
through both standard DSC and TMDSC was used to detect the underlying heat capacity and reveal the phase transitions of the
silk–metallic salts system. Results show that K+ metallic salts play the role of plasticizer in silk fibroin proteins, which reduces the glass transition (Tg) of the pure silk protein and negatively affects its structural thermal stability. On the other hand, Ca2+ metallic salts act as an anti-plasticizer, and increase the glass transition and the thermal stability of the silk protein
structure. This indicates that the thermal analysis methods offer a new pathway to study protein–metallic ion systems, yielding
very fruitful information for the study of protein structures in the future.