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In this paper, we demonstrate the coupling of synchrotron small angle X-ray scattering (SAXS) to asymmetrical flow-field flow fractionation (AF4) for protein characterization. To the best of our knowledge, this is the first time AF4 is successfully coupled to a synchrotron for on-line measurements on proteins. This coupling has potentially high impact, as it opens the possibility to characterize individual constituents of sensitive and/or complex samples, not suited for separation using other techniques, and for low electron density samples where high X-ray flux is required, e.g., biomolecules and biologics. AF4 fractionates complex samples in native or close to native environment, with low shear forces and system surface area. Many orders of magnitude in size can be fractionated in one measurement, without having to reconfigure the experimental setup. We report AF4 fractionations with correlated UV and statistically adequate SAXS data of bovine serum albumin and a monoclonal antibody and evaluate SAXS data recorded for the two protein systems., (© 2023. The Author(s).)

Transient biomolecular interactions play crucial roles in many cellular signaling and regulation processes. However, deciphering the structure of these assemblies is challenging owing to the difficulties in isolating complexes from the individual partners. The additive nature of small-angle X-ray scattering (SAXS) data allows for probing the species present in these mixtures, but decomposition into structural and thermodynamic information is difficult. We present a chemometric approach enabling the decomposition of titration SAXS data into species-specific information. Using extensive synthetic SAXS data, we demonstrate that robust decomposition can be achieved for titrations with a maximum fraction of complex of 0.5 that can be extended to 0.3 when two orthogonal titrations are simultaneously analyzed. The effect of the structural features, titration points, relative concentrations, and noise are thoroughly analyzed. The validation of the strategy with experimental data highlights the power of the approach to provide unique insights into this family of biomolecular assemblies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Formation of amyloids is the hallmark of several neurodegenerative pathologies. Structural investigation of these complex transformation processes poses significant experimental challenges due to the co-existence of multiple species. The additive nature of small-angle X-ray scattering (SAXS) data allows for probing the evolution of these mixtures of oligomeric states, but the decomposition of SAXS data into species-specific spectra and relative concentrations is burdened by ambiguity. We present an objective SAXS data decomposition method by adapting the multivariate curve resolution alternating least squares (MCR-ALS) chemometric method. The approach enables rigorous and robust decomposition of synchrotron SAXS data by simultaneously introducing these data in different representations that emphasize molecular changes at different time and structural resolution ranges. The approach has allowed the study of fibrillogenic forms of insulin and the familial mutant E46K of α-synuclein, and is generally applicable to any macromolecular mixture that can be probed by SAXS., (Copyright © 2016 Elsevier Ltd. All rights reserved.)