Compensation of Strong Water Absorption in Infrared Spectroscopy Reveals the Secondary Structure of Proteins in Dilute Solutions
Abstract
Infrared (IR) absorption spectroscopy is a powerful tool that can quantify 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 an adaptive solvent absorption compensation (SAC) approach 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 post-processing show good linearity from 100 mg/mL 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 °C for 18 hours. 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.
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