Abstract
Ultrafast two-dimensional infrared (2D-IR) spectra can now be obtained in a matter of seconds, opening up the possibility of high-throughput screening applications of relevance to the biomedical and pharmaceutical sectors. Determining quantitative information from 2D-IR spectra recorded on different samples and different instruments is however made difficult by variations in beam alignment, laser intensity, and sample conditions. Recently, we demonstrated that 2D-IR spectroscopy of the protein amide I band can be performed in aqueous (H2O) rather than deuterated (D2O) solvents, and we now report a method that uses the magnitude of the associated thermal response of H2O 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.
Highlights
Ultrafast two-dimensional infrared (2D-IR) spectra can be obtained in a matter of seconds, opening up the possibility of high-throughput screening applications of relevance to the biomedical and pharmaceutical sectors
Ultrafast 2D-IR spectroscopy is established as a powerful tool for interrogating the structure and dynamics of molecules in the solution phase and has played a significant role in developing our understanding of biomolecular systems, such as proteins and nucleic acids.[1−9] Recent developments in laser technology and mid-infrared pulse shaping[10−13] have provided scope for 2D-IR to be applied in a more analytical manner, for example, in high-throughput measurements for rapid screening of multiple biomolecule−drug combinations.[14]
We demonstrated recently that 2D-IR spectroscopy can be used to measure the amide I vibrational band of proteins in aqueous (H2O) solutions at submillimolar concentrations.[15]
Summary
Ultrafast two-dimensional infrared (2D-IR) spectra can be obtained in a matter of seconds, opening up the possibility of high-throughput screening applications of relevance to the biomedical and pharmaceutical sectors. The magnitudes of the amide I and thermal H2O signals are influenced in an identical manner by both sample-related (concentration, path length) and instrumental factors (laser intensity, beam quality, beam alignment).[1,16,17] the ratio of the magnitudes of the thermal response of water and the resonant amide I response of BSA in each measurement should be constant for a given BSA
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