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.

A guide to studying protein aggregation

“…The absolute molecular weight determination of SEC‐MALS does not depend on size/mass calibration and is thus more reliable compared to SEC alone [134,137]. However, SEC‐MALS is not suited for unpurified samples and is not sufficient to resolve mutants and variants with the same or very similar mass [135]. For analytes that cannot be eluted on SEC, ion‐exchange chromatography (IEX) can be coupled with MALS since IEX has a higher resolution compared with SEC and allows precise analysis of particles of comparable sizes [141].…”

“…The coupling of size exclusion chromatography (SEC) to multiple angle light scattering (MALS), known as SEC‐MALS, enables the characterization of molecular weight of different protein species within a sample [134–139]. SEC separates particles based on their size: bigger species elute faster than smaller ones (Fig.…”

“…2E) [134]. MALS in turn determines the absolute molecular weight of each eluted species via measuring the light intensity scattered by particles at multiple angles using MALS detector [134–136]. Since the function of the scattered light intensity with the scattering angle is proportional to the particle’s molecular weight and concentration, this allows the calculation of the eluting particle’s molecular weight once the concentration is accurately determined using a UV or differential refractive index (RI) detector [140].…”

How to Characterize the Protein Structure and Polymer Conformation in Protein‐Polymer Conjugates – a Perspective

“…Consequently, if the analyte of interest is different in shape or compactness or interacts with the column material, retention might be influenced to a large extent and absolute molar mass determination based on the reference becomes invalid. [64,65,67,68] SEC is widely used in the analysis of macromolecules, especially proteins and polymers. Proteins vary in shape and their Stokes radii do not correlate directly with their molar mass.…”

“…This method relies on the Rayleigh theory which describes the elastic scattering of light and the correlation between molar mass and size. [66,68,69] Obtained data can be analyzed for example by using a Zimm plot representation to determine the molar masses of the individual sample components (Figure 5c). However, a major challenge in the calculation of molar masses from MALS represents the determination of the refractive index increment dn/dc.…”