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Pushing the detection limit of infrared spectroscopy for structural analysis of dilute protein samples.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2014 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 139, no 21, 5393-5399 p.Article in journal (Refereed) Published
Abstract [en]

Fourier-transform infrared spectroscopy is a powerful and versatile tool to investigate the structure and dynamics of proteins in solution. The intrinsically low extinction coefficient of the amide I mode, the main structure-related oscillator, together with the high infrared absorptivity of aqueous media, requires that proteins are studied at high concentrations (>10 mg L(-1)). This may represent a challenge in the study of aggregation-prone proteins and peptides, and questions the significance of structural data obtained for proteins physiologically existing at much lower concentrations. Here we describe the development of a simple experimental approach that increases the detection limit of protein structure analysis by infrared spectroscopy. Our approach relies on custom-made filters to isolate the amide I region (1700-1600 cm(-1)) from irrelevant spectral regions. The sensitivity of the instrument is then increased by background attenuation, an approach consisting in the use of a neutral density filter, such as a non-scattering metal grid, to attentuate the intensity of the background spectrum. When the filters and grid are combined, a 2.4-fold improvement in the noise level can be obtained. We have successfully tested this approach using a highly diluted solution of pyruvate kinase in deuterated medium (0.2% w/v), and found that it provides spectra of a quality comparable to those recorded with a 10-fold higher protein concentration.

Place, publisher, year, edition, pages
2014. Vol. 139, no 21, 5393-5399 p.
Keyword [en]
Infrared spectroscopy, FTIR spectroscopy, protein, amide I, secondary structure
National Category
Analytical Chemistry
URN: urn:nbn:se:su:diva-108964DOI: 10.1039/c4an00918eISI: 000343003700012PubMedID: 25163493OAI: diva2:761792
Available from: 2014-11-07 Created: 2014-11-07 Last updated: 2014-11-17Bibliographically approved

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Baldassarre, MaurizioBarth, Andreas
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