Compensation of Strong Water Absorption in Infrared Spectroscopy Reveals the Secondary Structure of Proteins in Dilute Solutions

Infrared (IR) absorption spectroscopy is a powerful tool that can quanti complex biomolecules and their structural conformations. However, conventional approaches to protein analysis in aqueous solutions have been significantly challenged because the strong IR absorption of water overwhelms the limited dynamic range of the detection system and thus allows only a very short path length and a limited concentration sensitivity. Here, we demonstrate a solvent absorption compensation (SAC) approach that can improve the concentration sensitivity and extend the available path length by distinguishing the analyte signal over the full dynamic range at each wavelength. Absorption spectra without any postprocessing show good linearity from 100 to 0.1 mg/mL protein concentration, allowing a >100 times enhanced signal-to-noise ratio in the amide I band compared to the non-SAC results. We apply this method to in situ investigate the isothermal kinetics of insulin fibrillation at two clinical concentrations at 74 degrees C for 18 h. Simultaneous monitoring of both reactants (native forms) and products (fibrils) allows quantitative discussion of the detailed fibrillation mechanisms, which are not accessible with other single modality measurements. This simple optical technique can be applied to other absorption spectroscopies of analytes in strongly absorbing solvents, allowing for enhanced sensitivity without changing the detection system.

Automated summary

In this study, a solvent absorption compensation (SAC) approach is proposed to enhance the concentration sensitivity and extend the available path length for protein analysis using infrared (IR) absorption spectroscopy in aqueous solutions. By applying this method, absorption spectra show good linearity and enhanced signal-to-noise ratio, allowing for more accurate investigation of protein structural conformations and reactions. The SAC method can also be applied to other absorption spectroscopies of analytes in strongly absorbing solvents for improved sensitivity without changing the detection system.