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Pushing the detecti...
Pushing the detection limit of infrared spectroscopy for structural analysis of dilute protein samples.
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- Baldassarre, Maurizio (author)
- Stockholms universitet,Institutionen för biokemi och biofysik
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- Barth, Andreas (author)
- Stockholms universitet,Institutionen för biokemi och biofysik
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(creator_code:org_t)
- 2014
- 2014
- English.
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In: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 139:21, s. 5393-5399
- Related links:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Subject headings
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- 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.
Subject headings
- NATURVETENSKAP -- Kemi -- Analytisk kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences -- Analytical Chemistry (hsv//eng)
Keyword
- Infrared spectroscopy
- FTIR spectroscopy
- protein
- amide I
- secondary structure
Publication and Content Type
- ref (subject category)
- art (subject category)
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