title: Pushing the detection limit of infrared spectroscopy for structural analysis of dilute protein samples.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden. maurizio.baldassarre@dbb.su.se andreas.barth@dbb.su.se.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Baldassarre
authors.forename: Andreas
authors.initials: A
authors.lastname: Barth
mesh_terms: D002482:Cellulose / Q000737:chemistry; D005857:Germanium / Q000737:chemistry; D057230:Limit of Detection; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D013055:Spectrophotometry, Infrared / Q000379:methods*
chemical_list: D011506:Proteins; D005857:Germanium; D002482:Cellulose