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Infrared absorbance spectroscopy of aqueous proteins : comparison of transmission and ATR data collection and analysis for secondary structure fitting
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Corujo, Marco Pinto, Sklepari, Meropi, Ang, Dale L., Millichip, Mark, Reason, Andrew, Goodchild, Sophia C., Wormell, Paul, Amarasinghe, Don Praveen, Lindo, Viv, Chmel, Nikola Paul and Rodger, Alison (2018) Infrared absorbance spectroscopy of aqueous proteins : comparison of transmission and ATR data collection and analysis for secondary structure fitting. Chirality, 30 (8). pp. 957-965. doi:10.1002/chir.23002
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WRAP-infrared-absorbance-spectroscopy-aqueous-proteins-analysis-Corujo-2018.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution 4.0. Download (658Kb) | Preview |
Official URL: https://doi.org/10.1002/chir.23002
Abstract
Attenuated total reflectance (ATR) infrared absorbance spectroscopy of proteins in aqueous solution is much easier to perform than transmission spectroscopy, where short path‐length cells need to be assembled reproducibly. However, the shape of the resulting ATR infrared spectrum varies with the refractive index of the sample and the instrument configuration. Refractive index in turn depends on the absorbance of the sample. In this work, it is shown that a room temperature triglycine sulfate detector and a ZnSe ATR unit can be used to collect reproducible spectra of proteins. A simple method for transforming the protein ATR spectrum into the shape of the transmission spectrum is also given, which proceeds by approximating a Kramers‐Krönig–determined refractive index of water as a sum of four linear components across the amide I and II regions. The light intensity at the crystal surface (with 45° incidence) and its rate of decay away from the surface is determined as a function of the wave number–dependent refractive index as well as the decay of the evanescent wave from the surface. The result is a single correction factor at each wave number. The spectra were normalized to a maximum of 1 between 1600 cm−1 and 1700 cm−1 and a self‐organizing map secondary structure fitting algorithm, SOMSpec, applied using the BioTools reference set. The resulting secondary structure estimates are encouraging for the future of ATR spectroscopy for biopharmaceutical characterization and quality control applications.
| Item Type: | Journal Article | |||||||||||||||
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| Subjects: | Q Science > QD Chemistry | |||||||||||||||
| Divisions: | Faculty of Science > Chemistry Faculty of Science > Molecular Organisation and Assembly in Cells (MOAC) |
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| SWORD Depositor: | Library Publications Router | |||||||||||||||
| Library of Congress Subject Headings (LCSH): | Absorbance scale (Spectroscopy), Absorption spectra, Proteins | |||||||||||||||
| Journal or Publication Title: | Chirality | |||||||||||||||
| Publisher: | Wiley | |||||||||||||||
| ISSN: | 0899-0042 | |||||||||||||||
| Official Date: | August 2018 | |||||||||||||||
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| Volume: | 30 | |||||||||||||||
| Number: | 8 | |||||||||||||||
| Page Range: | pp. 957-965 | |||||||||||||||
| DOI: | 10.1002/chir.23002 | |||||||||||||||
| Status: | Peer Reviewed | |||||||||||||||
| Publication Status: | Published | |||||||||||||||
| Access rights to Published version: | Open Access | |||||||||||||||
| RIOXX Funder/Project Grant: |
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