title: Methods for analysis of size-exclusion chromatography-small-angle X-ray scattering and reconstruction of protein scattering.
abstract: Size-exclusion chromatography in line with small-angle X-ray scattering (SEC-SAXS) has emerged as an important method for investigation of heterogeneous and self-associating systems, but presents specific challenges for data processing including buffer subtraction and analysis of overlapping peaks. This paper presents novel methods based on singular value decomposition (SVD) and Guinier-optimized linear combination (LC) to facilitate analysis of SEC-SAXS data sets and high-quality reconstruction of protein scattering directly from peak regions. It is shown that Guinier-optimized buffer subtraction can reduce common subtraction artifacts and that Guinier-optimized linear combination of significant SVD basis components improves signal-to-noise and allows reconstruction of protein scattering, even in the absence of matching buffer regions. In test cases with conventional SAXS data sets for cytochrome c and SEC-SAXS data sets for the small GTPase Arf6 and the Arf GTPase exchange factors Grp1 and cytohesin-1, SVD-LC consistently provided higher quality reconstruction of protein scattering than either direct or Guinier-optimized buffer subtraction. These methods have been implemented in the context of a Python-extensible Mac OS X application known asData Evaluation and Likelihood Analysis(DELA), which provides convenient tools for data-set selection, beam intensity normalization, SVD, and other relevant processing and analytical procedures, as well as automated Python scripts for common SAXS analyses and Guinier-optimized reconstruction of protein scattering.
authors: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA ; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors: Andrew W
authors: AW
authors: Malaby
authors: The Biophysics Collaborative Access Team (BioCAT), Department of Biological Chemical and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA.
authors: Srinivas
authors: S
authors: Chakravarthy
authors: The Biophysics Collaborative Access Team (BioCAT), Department of Biological Chemical and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA.
authors: Thomas C
authors: TC
authors: Irving
authors: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors: Sagar V
authors: SV
authors: Kathuria
authors: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors: Osman
authors: O
authors: Bilsel
authors: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA ; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors: David G
authors: DG
authors: Lambright
keywords: Guinier optimization; linear combination; singular value decomposition; size-exclusion chromatography; small-angle X-ray scattering