US-SOMO HPLC-SAXS module: dealing with capillary fouling and extraction of pure component patterns from poorly resolved SEC-SAXS data.

Emre Brookes,Patrice Vachette,Mattia Rocco,Javier Pérez

doi:10.1107/s1600576716011201

Abstract

Size-exclusion chromatography coupled with SAXS (small-angle X-ray scattering), often performed using a flow-through capillary, should allow direct collection of monodisperse sample data. However, capillary fouling issues and non-baseline-resolved peaks can hamper its efficacy. The UltraScan solution modeler (US-SOMO) HPLC-SAXS (high-performance liquid chromatography coupled with SAXS) module provides a comprehensive framework to analyze such data, starting with a simple linear baseline correction and symmetrical Gaussian decomposition tools [Brookes, Pérez, Cardinali, Profumo, Vachette & Rocco (2013 ▸). J. Appl. Cryst.46, 1823-1833]. In addition to several new features, substantial improvements to both routines have now been implemented, comprising the evaluation of outcomes by advanced statistical tools. The novel integral baseline-correction procedure is based on the more sound assumption that the effect of capillary fouling on scattering increases monotonically with the intensity scattered by the material within the X-ray beam. Overlapping peaks, often skewed because of sample interaction with the column matrix, can now be accurately decomposed using non-symmetrical modified Gaussian functions. As an example, the case of a polydisperse solution of aldolase is analyzed: from heavily convoluted peaks, individual SAXS profiles of tetramers, octamers and dodecamers are extracted and reliably modeled.

Highlights

Multi-resolution approaches for the structural characterization of complex macromolecular samples, such as in the presence of segmental/extended flexibility, or when supramolecular entities form in solution, are becoming increasingly used
Spurred by the need to analyze a complex fibrinogen sample, a dedicated HPLC-Small-angle X-ray scattering (SAXS) module was developed by Brookes et al (2013) as a part of the small-angle scattering (SAS) section of the data analysis and simulation open source platform UltraScan solution modeler (USSOMO; http://somo.uthscsa.edu/; Brookes, Demeler & Rocco, 2010; Brookes, Demeler et al, 2010; Rocco & Brookes, 2014)
In contrast with simpler programs that deal mainly with the automation of the repetitive tasks involved in analyzing the single different frames coming from a SEC-SAXS experiment (e.g. Shkumatov & Strelkov, 2015), the UltraScan solution modeler (US-SOMO) SAS and HPLC-SAXS modules were developed from their inception with the aim of providing advanced tools to deal with all aspects involved, from primary data treatment to the decomposition of unresolved components, and the comparison with model curves derived from high-resolution data

Summary

Introduction

Multi-resolution approaches for the structural characterization of complex macromolecular samples, such as in the presence of segmental/extended flexibility, or when supramolecular entities form in solution, are becoming increasingly used (see e.g. Ward et al, 2013). Shkumatov & Strelkov, 2015), the US-SOMO SAS and HPLC-SAXS modules were developed from their inception with the aim of providing advanced tools to deal with all aspects involved, from primary data treatment to the decomposition of unresolved components, and the comparison with model curves derived from high-resolution data. This last step is currently based on the embedded well known programs

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Results

Conclusion