Factors Influencing the Characterization of Gluten Proteins by Size‐Exclusion Chromatography and Multiangle Laser Light Scattering (SEC‐MALLS)

Abstract

ABSTRACTThe use of multiangle laser light scattering (MALLS) in conjunction with size exclusion chromatography (SEC) was investigated for characterizing wheat proteins. Four solvent systems including 50% acetonitrile and 0.1% trifluoroacetic acid, 50 mM sodium phosphate (NaPhos) pH 2.5, 500 mM acetic acid, and 50 mM NaPhos pH 7.0 + 1% SDS were evaluated for protein extraction as well as for use as SEC mobile phases for MALLS analysis. The dn/dc values for wheat proteins were measured in each solvent. Except for the SDS‐based mobile phase, gluten proteins showed dn/dc values of 0.16–0.20 that were similar to values reported for other proteins. When analyzed in the SDS solvent, gluten proteins showed dn/dc values of 0.32, similar to that found for other SDS‐protein complexes. While all solvents showed similar resolution when used as mobile phases in SEC analysis, the SDS solvent extracted the most protein (≈82%) in the unreduced form. This solvent system also displayed no concentrationdependent or electrostatic effects during MALLS analysis. SDS‐soluble and insoluble protein complexes were characterized by MALLS. Mw distributions of 8.1 × 107 Da were found for the SDS‐insoluble protein complexes. The effect of the column void volume was also examined as was data analysis parameters such as fitting method and peak placement.