Abstract
Biological small-angle X-ray scattering (BioSAXS) is a powerful technique to determine the solution structure, particle size, shape and surface-to-volume ratio of macromolecules. However, a drawback is that the sample needs to be monodisperse. To ensure this, size-exclusion chromatography (SEC) has been implemented on many BioSAXS beamlines. Here, the integration of ion-exchange chromatography (IEC) using both continuous linear and step gradients on a beamline is described. Background subtraction for continuous gradients by shifting a reference measurement and two different approaches for step gradients, which are based on interpolating between two background measurements, are discussed. The results presented here serve as a proof of principle for online IEC and subsequent data treatment.
Highlights
Biological small-angle X-ray scattering (BioSAXS) can reveal solution structures in terms of the average particle size and shape of biological macromolecules, as well as information on the surface-to-volume ratio (Graewert & Svergun, 2013; Kikhney & Svergun, 2015; Putnam et al, 2007; Jacques & Trewhella, 2010)
To the salt gradient in ionexchange chromatography (IEC)–SAXS used here, the background signal in differential ultracentrifugation-coupled SAXS changes owing to a sucrose gradient
We found that the regions used by Hynson and coworkers (0.11–0.5 and 1.5–2.5 nmÀ1 for low-q and mid-q regions, respectively) suited well: the low-q region reflects the correct overall size of the protein, whereas most of the characteristic features of bovine serum albumin (BSA) are present in the mid-q region
Summary
Biological small-angle X-ray scattering (BioSAXS) can reveal solution structures in terms of the average particle size and shape of biological macromolecules, as well as information on the surface-to-volume ratio (Graewert & Svergun, 2013; Kikhney & Svergun, 2015; Putnam et al, 2007; Jacques & Trewhella, 2010). The combination of online size-exclusion chromatography (SEC) and SAXS has been implemented directly on beamlines in order to overcome this obstacle, to ensure data quality and to make this technique more accessible for increasingly difficult samples (Lambright et al, 2013; Round et al, 2013; David & Perez, 2009; Graewert et al, 2015; Mathew et al, 2004; Watanabe & Inoko, 2009; Acerbo et al, 2015; Grant et al, 2011) Additional techniques, such as time-resolved (TR) SAXS experiments using desalting columns for quick buffer exchanges (Jensen et al, 2010) and differential ultracentrifugation, have been successfully coupled with SAXS (Hynson et al, 2015). We found that the regions used by Hynson and coworkers (0.11–0.5 and 1.5–2.5 nmÀ1 for low-q and mid-q regions, respectively) suited well: the low-q region reflects the correct overall size of the protein, whereas most of the characteristic features of BSA are present in the mid-q region (see, for example, Fig. 5d)
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