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

Samantha H. Rutherford, Gregory M. Greetham, Anthony W. Parker, Alison Nordon, Matthew J. Baker, Neil T. Hunt

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)
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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.

Original languageEnglish
Article number205102
Number of pages11
JournalJournal of Chemical Physics
Issue number20
Early online date7 Nov 2022
Publication statusPublished - 22 Nov 2022


  • measurement
  • proteins
  • H20
  • 2D-IR spectroscopy
  • pre-processing steps
  • protein library


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