2D-Infrared Spectroscopy of Proteins in Water: Using the Solvent Thermal Response as an Internal Standard
Samantha Hume, Gregory M. Greetham, Paul M. Donaldson, Michael Towrie, Anthony W. Parker, Matthew J. Baker, Neil T. Hunt
Abstract
O as an internal normalization standard for 2D-IR spectra. Using the water response, which is temporally separated from the protein signal, to normalize the spectra allows significant reduction of the impact of measurement-to-measurement fluctuations on the data. We demonstrate that this normalization method enables creation of calibration curves for measurement of absolute protein concentrations and facilitates reproducible difference spectroscopy methodologies. These advances make significant progress toward the robust data handling strategies that will be essential for the realization of automated spectral analysis tools for large scale 2D-IR screening studies of protein-containing solutions and biofluids.