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Realizing the AF4-UV-SAXS on-line coupling on protein and antibodies using high flux synchrotron radiation at the CoSAXS beamline, MAX IV.

ABSTRACT: 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.

SUBMITTER: Bolinsson H

PROVIDER: S-EPMC10558385 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Realizing the AF4-UV-SAXS on-line coupling on protein and antibodies using high flux synchrotron radiation at the CoSAXS beamline, MAX IV.

Bolinsson Hans H Söderberg Christopher C Herranz-Trillo Fátima F Wahlgren Marie M Nilsson Lars L

Analytical and bioanalytical chemistry 20230812 25

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 te ...[more]

PMID: 37572213

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Project description:Characterization of the mechanical response of polymers and composite materials relies heavily on the macroscopic stress-strain response in uniaxial tensile configurations. To provide representative information, the deformation process must be homogeneous within the gauge length, which is a condition that is rarely achieved due to stress concentration or inhomogeneities within the specimen. In this work, the development of a biaxial mechanical testing device at the CoSAXS beamline at MAX IV Laboratory is presented. The design facilitates simultaneous measurement of small- and wide-angle X-ray scattering (SAXS/WAXS), allowing assessment of the microstructural configuration before, after and during the continuous deformation process at multiple length scales. The construction also supports multiple deformation conditions, while guaranteeing stability even at high loads. Furthermore, the mechanical experiments can be complemented with spatially resolved mesoscopic surface deformation measurements using 3D-surface digital image correlation (DIC). Polycarbonate (PC) was used to demonstrate the varied material response to multi-axial deformation, as PC is isotropic with a high glass transition temperature (∼150°) and high strength. As a result, a clear correlation between full-field methods and the microstructural information determined from WAXS measurements is demonstrated. When a uniaxial load is applied, homogeneous strain regions could be observed extending perpendicular to the applied load. When a secondary axial load was added (biaxial mode), it was observed that high strain domains were created near the centre of the sample and at the boundaries after yield. With increased strain, the deformation in the main deformation direction also increases. Mechanical reliability was demonstrated by carrying out static loading of polyacrylonitrile-based carbon fibre (CF) bundles. As a result, the nonlinear stiffening behaviour typically observed in CFs was seen, while no evidence of the creation of new voids during loading was observed. The results support the reliability and broad applicability of the developed technique.

Project description:Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J. Phys. Conf. Ser. 425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser. 493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006]. FMX, the micro-focusing Frontier MX beamline in sector 17-ID-2 at NSLS-II, covers a 5-30 keV photon energy range and delivers a flux of 4.0 × 1012 photons s-1 at 1 Å into a 1 µm × 1.5 µm to 10 µm × 10 µm (V × H) variable focus, expected to reach 5 × 1012 photons s-1 at final storage-ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods - serial crystallography on micrometre-sized crystals, raster optimization of diffraction from inhomogeneous crystals, high-resolution data collection from large-unit-cell crystals, room-temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample-screening and ligand-binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample-delivery methods have been implemented, such as an ultra-high-speed high-precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad. 25, 1362-1370], new microcrystal-optimized micromesh well sample holders [Guo et al. (2018). IUCrJ, 5, 238-246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun. 5, 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult-to-crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.

Dataset Information

Realizing the AF4-UV-SAXS on-line coupling on protein and antibodies using high flux synchrotron radiation at the CoSAXS beamline, MAX IV.

Publications

Realizing the AF4-UV-SAXS on-line coupling on protein and antibodies using high flux synchrotron radiation at the CoSAXS beamline, MAX IV.

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