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

Rutherford, Samantha H. and Greetham, Gregory M. and Parker, Anthony W. and Nordon, Alison and Baker, Matthew J. and Hunt, Neil T. (2022) Measuring proteins in H2O using 2D-IR spectroscopy : pre-processing steps and applications toward a protein library. Journal of Chemical Physics, 157 (20). 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.