Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Abstract

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Structural and Dynamic Insights Into α-Synuclein Dimer Conformations

“…AIEX-MALS has proved to be indispensable in reporting on the ionic strength, the molecular mass and the hydrodynamic radius of each elutant, independently (26,27). In AIEX-MALS experiments of WT αSyn, we identified a single elution peak with a molecular mass larger than expected from the monomer (> 14.4 kDa).…”

“…αSyn does not have a well-defined structure, which is why it may be hard to connect its band position in PAGE to the molecular mass. In MALS, the molecular mass is calculated from two direct optical readouts, namely the protein light absorption and the light scattering intensity, used in the Rayleigh scattering formula (26,27,30). The light scattering intensity in our experiments was independent of the shape of the scattering proteins, since the incident light wavelength, 658.9 nm, is much larger than the sizes of the scattering protein molecules.…”

“…AIEX allows probing different elution peaks in chromatography based on their ionic strength values. Coupling analytical AIEX chromtography to multi angle light scattering (MALS; see Experimental Procedures) allows probing different elution peaks for their molecular mass directly, rather than via transformation from its hydrodynamic radius (RH) or its radius of gyration (RG) (26,27,30). In addition to the MALS system, a quasi-elastic light scattering module also allowed measuring dynamic light scattering (DLS) curves, from which the values of RH were retrieved, in addition to and independent of the molecular mass estimation.…”

“…1, B, dotted lines). We tried to gain information regarding the validity of the monomer-dimer mixture hypothesis by coupling size exclusion chromatography (SEC; superdex 75) to MALS (26). In the SEC-MALS chromatogram we identified two elution peaks (Fig.…”

“…Knowing the dissociation constant of the noncovalent αSyn dimer would help define how to study the mechanism of the dimer formation. In this work we use anion exchange chromatography (AIEX) coupled to multi angle light scattering (MALS) (26,27) to objectively and directly link each species eluted in a chromatographic run of αSyn to its average molecular mass. We show that the ensemble of freshly-prepared recombinant αSyn exists in an equilibrium between monomers and compact dimers with a large dimer fraction when at micromolar concentration.…”