Fourier transform infrared (FTIR) spectroscopy is one of the widely used vibrational spectroscopic methods in protein structural analysis. The protein solution sample loaded in demountable CaF2 liquid cell presents a challenge and is limited to high concentrations. Some researchers attempted the simpler solid-film sampling method for the collection of protein FTIR spectra. In this study, the solid-film sampling FTIR method was studied in detail. The secondary structure components of some globular proteins were determined by this sampling method, and the results were consistent with those data determined by the traditional solution sampling FTIR method and X-ray crystallography, indicating that this sampling method is feasible and efficient for the structural characterization of proteins. Furthermore, much lower protein concentrations (~0.5 mg/mL) were needed to obtain high-quality FTIR spectra, which expands the application of FTIR spectroscopy to almost the same concentration range used for circular dichroism and fluorescence spectroscopy, making comparisons among three commonly used techniques possible in protein studies. Graphical Abstract ᅟ.
Keywords: FTIR spectroscopy; Protein concentration; Protein secondary structural composition; Protein structure.