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...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Cambridge University Press
2020-01-01
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| Series: | QRB Discovery |
| Subjects: | |
| Online Access: | https://www.cambridge.org/core/product/identifier/S2633289220000113/type/journal_article |
| _version_ | 1811155758111260672 |
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| author | Alison Rodger Michael J. Steel Sophia C. Goodchild Nikola P. Chmel Andrew Reason |
| author_facet | Alison Rodger Michael J. Steel Sophia C. Goodchild Nikola P. Chmel Andrew Reason |
| author_sort | Alison Rodger |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T04:38:55Z |
| format | Article |
| id | doaj.art-b9d06e1b68614cf48ff8ea59962aa8fd |
| institution | Directory Open Access Journal |
| issn | 2633-2892 |
| language | English |
| last_indexed | 2024-04-10T04:38:55Z |
| publishDate | 2020-01-01 |
| publisher | Cambridge University Press |
| record_format | Article |
| series | QRB Discovery |
| spelling | doaj.art-b9d06e1b68614cf48ff8ea59962aa8fd2023-03-09T12:43:33ZengCambridge University PressQRB Discovery2633-28922020-01-01110.1017/qrd.2020.11Transformation of aqueous protein attenuated total reflectance infra-red absorbance spectroscopy to transmissionAlison Rodger0https://orcid.org/0000-0002-7111-3024Michael J. Steel1https://orcid.org/0000-0002-6789-0381Sophia C. Goodchild2https://orcid.org/0000-0002-5091-7576Nikola P. Chmel3https://orcid.org/0000-0002-2882-5253Andrew Reason4Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, AustraliaDepartment of Physics and Astronomy, Macquarie University, Sydney, NSW 2109, AustraliaDepartment of Molecular Sciences, Macquarie University, Sydney, NSW 2109, AustraliaDepartment of Chemistry, University of Warwick, Coventry CV4 7AL, UKBioPharmaSpec Ltd., Lido Medical Centre, St. Saviour, Jersey JE2 7LA, UKInfrared (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.https://www.cambridge.org/core/product/identifier/S2633289220000113/type/journal_articleAttenuated total reflectanceinfrared spectroscopyproteinstructuretransmissionwater |
| spellingShingle | Alison Rodger Michael J. Steel Sophia C. Goodchild Nikola P. Chmel Andrew Reason Transformation of aqueous protein attenuated total reflectance infra-red absorbance spectroscopy to transmission QRB Discovery Attenuated total reflectance infrared spectroscopy protein structure transmission water |
| title | Transformation of aqueous protein attenuated total reflectance infra-red absorbance spectroscopy to transmission |
| title_full | Transformation of aqueous protein attenuated total reflectance infra-red absorbance spectroscopy to transmission |
| title_fullStr | Transformation of aqueous protein attenuated total reflectance infra-red absorbance spectroscopy to transmission |
| title_full_unstemmed | Transformation of aqueous protein attenuated total reflectance infra-red absorbance spectroscopy to transmission |
| title_short | Transformation of aqueous protein attenuated total reflectance infra-red absorbance spectroscopy to transmission |
| title_sort | transformation of aqueous protein attenuated total reflectance infra red absorbance spectroscopy to transmission |
| topic | Attenuated total reflectance infrared spectroscopy protein structure transmission water |
| url | https://www.cambridge.org/core/product/identifier/S2633289220000113/type/journal_article |
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