Analysis of Oligomeric and Glycosylated Proteins by Size-Exclusion Chromatography Coupled with Multiangle Light Scattering.

Abstract

Analytical size-exclusion chromatography (SEC) is a powerful technique that separates proteins based on their hydrodynamic radii. This approach can provide some rudimentary information about the molecular weight of proteins, but results are also influenced by the in-solution protein conformation and hydrophobicity. SEC also can be affected by nonspecific interactions with the column matrix that influence protein separation. Light scattering (LS) is an absolute and highly accurate measurement of protein molecular weight. Coupling analytical size-exclusion chromatography with multiangle light scattering (SEC-MALS) yields a more robust and accurate method for determining multiple biophysical parameters of proteins while avoiding SEC artifacts. This union of two techniques can help determine the absolute molecular stoichiometry, homo- and heteroassociation of sample components, the nature of protein conjugates, and the molar mass of single molecules and multisubunit complexes. In this chapter, we provide several examples of analysis of glycosylated protein conjugates to showcase the power of SEC-MALS.