Real-time in-situ quantification of protein secondary structures in aqueous solution based on ATR-FTIR subtraction spectrum

Abstract Changes occur in protein secondary structures during food processing, pharmaceutical research, and enzymatic reactions, etc. To date, most studies to quantify the protein secondary structures in aqueous solution have just involved semi-quantitative analysis and cannot achieve real-time measurements. In this study, attenuated total internal reflectance Fourier transform infrared spectroscopy was used to analyze the protein secondary structures, and buffer solution subtraction was used to minimize the interference of water and other solvent molecules. A novel calculation and correction method including acquisition parameters, subtraction spectrum criteria, second derivative spectra, Fourier self-deconvolution (FSD), peak fitting and peak assignment, was developed and applied to the amide I band of buffer-subtraction spectrum for optimization. It is found that the calculation and correction method achieved good reproducibility and high accuracy with the protein’s secondary structure results of 10 replicates, with the maximum coefficient of variation of less than 0.2 and the maximum difference within the group of less than 0.03. Moreover, the quantitative results from this method were consistent with the results of circular dichroism experiments and molecular simulation. Thus, the proposed method is suitable for directly analyzing protein secondary structures in aqueous solutions with a wide range of concentrations and in different buffer systems.