Factors influencing the characterization of gluten proteins by size-exclusion chromatography and multiangle laser light scattering (SEC-MALLS)

The 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 (approximate to 82%) in the unreduced form. This solvent system also displayed no concentration-dependent or electrostatic effects during MALLS analysis. SDS-soluble and insoluble protein complexes were characterized by MALLS. M-w distributions of 8.1 x 10(7) 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.