Peptide Mapping with LC-HRMS and In Silico Tools for Evaluation of Potential Allergenicity of Commercially Available Whey Protein Hydrolysates

Hypoallergenic formulas containing hydrolyzed protein are intended for use by infants to prevent cow’s milk protein allergy. The degree of hydrolysis of epitopic areas determines the residual allergenicity of whey protein hydrolysates (WPHs). However, only amino-acid-based infant formulas (IFs) are considered entirely nonallergenic. The aim of this study was to investigate four commercially available WPHs with different degree of hydrolysis (H1–H4) for potential allergenicity, by applying LC-HRMS analysis of peptides, and using in silico tools to search for the immunoglobulin (IgE)-binding allergenic epitopes from the Immune Epitope Database. Additionally, the molecular weight distribution of proteins and peptides in the WPHs was measured by SE-UPLC. Based on the peptide coverage and peptide-length distribution profiles, the WPHs showed different extents of hydrolysis: extensively (H1 and H2), partially (H3), and slightly hydrolyzed (H4). Altogether, numerous peptides related to 46 specific IgE-binding epitopes from β-lactoglobulin and α-lactalbumin were found in all the WPHs, regardless of their extent of hydrolysis. Sequence-based identification of the specific peptide composition, with an application of in silico tools, is a reliable approach for discovering the potential allergenicity of protein hydrolysates for IFs.