Measuring proteins in H2O using 2D-IR spectroscopy: pre-processing steps and applications toward a protein library

Rutherford, Samantha H., Greetham, Gregory M., Parker, Anthony W., Nordon, Alison, Baker, Matthew orcid iconORCID: 0000-0003-2362-8581 and Hunt, Neil T. (2022) Measuring proteins in H2O using 2D-IR spectroscopy: pre-processing steps and applications toward a protein library. The Journal of Chemical Physics, 157 (20). p. 205102. ISSN 0021-9606

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The ability of two-dimensional infrared (2D-IR) spectroscopy to measure the amide I band of proteins in H2O rather than D2O-based solvents by evading the interfering water signals has enabled in vivo studies of proteins under physiological conditions and in biofluids. Future exploitation of 2D-IR in analytical settings, from diagnostics to protein screening, will, however, require comparisons between multiple datasets, necessitating control of data collection protocols to minimize measurement-to-measurement inconsistencies. Inspired by analytical spectroscopy applications in other disciplines, we describe a workflow for pre-processing 2D-IR data that aims to simplify spectral cross-comparisons. Our approach exploits the thermal water signal that is collected simultaneously with, but is temporally separated from the amide I response to guide custom baseline correction and spectral normalization strategies before combining them with Principal Component noise reduction tools. Case studies show that application of elements of the pre-processing workflow to previously published data enables improvements in quantification accuracy and detection limits. We subsequently apply the complete workflow in a new pilot study, testing the ability of a prototype library of 2D-IR spectra to quantify the four major protein constituents of blood serum in a single, label-free measurement. These advances show progress toward the robust data handling strategies that will be necessary for future applications of 2D-IR to pharmaceutical or biomedical problems.

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