Pushing the detection limit of infrared spectroscopy for structural analysis of dilute protein samples.
2014 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 139, no 21, 5393-5399 p.Article in journal (Refereed) Published
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.
Infrared spectroscopy, FTIR spectroscopy, protein, amide I, secondary structure
IdentifiersURN: urn:nbn:se:su:diva-108964DOI: 10.1039/c4an00918eISI: 000343003700012PubMedID: 25163493OAI: oai:DiVA.org:su-108964DiVA: diva2:761792