A Novel Size-Exclusion Chromatography Method for Subunit-Level Identification and Relative Quantification of Faba Bean Proteins

Abstract With increasing interest in faba bean as a plant-based protein crop, reliable methods to characterize protein composition are crucial for understanding structural and functional properties of faba bean-based raw materials and ingredients. A novel method combining size-exclusion high-performance liquid chromatography (SE-HPLC) with peak deconvolution was developed to separate and relatively quantify 7S and 11S globulins and other faba bean proteins at their monomer/subunit level. Peaks were assigned to specific protein fractions based on apparent molecular weight calibration, SDS-PAGE densitometry, and LC-MS/MS analysis. The method was validated using 7S and 11S globulin-enriched fractions and successfully applied to faba bean flours and protein isolates, confirming applicability across different ingredient purities (26.5–78.6% protein) and protein composition (0.83–1.40 7S/11S globulin ratio). It effectively resolves proteins with similar molecular weight, particularly 11S subunits and 7S proteolytic fragments. Overall, this high-throughput approach enables rapid and accurate identification and relative quantification of faba bean protein composition.