Transformation of aqueous protein attenuated total reflectance infra-red absorbance spectroscopy to transmission

Infrared (IR) spectroscopy is increasingly being used to probe the secondary structure of proteins, especially for high-concentration samples and biopharmaceuticals in complex formulation vehicles. However, the small path lengths required for aqueous protein transmission experiments, due to high wat...

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Bibliographic Details
Published in:QRB discovery 2020, Vol.1, p.e8-e8, Article e8
Main Authors: Rodger, Alison, Steel, Michael J, Goodchild, Sophia C, Chmel, Nikola P, Reason, Andrew
Format: Article
Language:English
Subjects:
Absorbance
Attenuated total reflectance
Concanavalin A
Experiments
Hemoglobin
Incidence angle
Infrared spectroscopy
Light
Lysozyme
Mercury cadmium telluride
protein
Proteins
Reflectance
Refractivity
Spectra
Spectrum analysis
structure
Transformations
transmission
water
Wavelengths
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Summary:Infrared (IR) spectroscopy is increasingly being used to probe the secondary structure of proteins, especially for high-concentration samples and biopharmaceuticals in complex formulation vehicles. However, the small path lengths required for aqueous protein transmission experiments, due to high water absorbance in the amide I region of the spectrum, means that the path length is not accurately known, so only the shape of the band is ever considered. This throws away a dimension of information. Attenuated total reflectance (ATR) IR spectroscopy is much easier to implement than transmission IR spectroscopy and, for a given instrument and sample, gives reproducible spectra. However, the ATR-absorbance spectrum varies with sample concentration and instrument configuration, and its wavenumber dependence differs significantly from that observed in transmission spectroscopy. In this paper, we determine, for the first time, how to transform water and aqueous protein ATR spectra into the corresponding transmission spectra with appropriate spectral shapes and intensities. The approach is illustrated by application to water, concanavalin A, haemoglobin and lysozyme. The transformation is only as good as the available water refractive index data. A hybrid of literature data provides the best results. The transformation also allows the angle of incidence of an ATR crystal to be determined. This opens the way to using both spectral shape and spectra intensity for protein structure fitting.
ISSN:2633-2892
2633-2892
DOI:10.1017/qrd.2020.11