Egg proteins are a good source of bioactive peptides. Previous work from our research group has demonstrated the potential of egg white hydrolysate (EWH) for enhancing adipogenic differentiation and insulin signalling in 3T3-F442A pre-adipocytes. EWH was prepared by a combination of thermolysin and pepsin. Here, in this study, we aimed to identify the responsible peptide(s) in EWH. EWH was fractionated stepwise by ultrafiltration, C18 Sep-Pack cartridge, cation-exchange chromatography, and reverse-phase chromatography. The two most active fractions were analyzed by LC–MS/MS and 42 peptides were identified. Eleven peptides were synthesized and WEKAFKDED, QAMPFRVTEQE, ERYPIL, and VFKGL from ovalbumin were validated with peroxisome proliferator-associated receptor gamma stimulatory activity in adipocytes. For the first time, adipogenic differentiating peptides were characterized from egg white proteins. This data is pivotal for future structure–function studies of adipogenic peptides.
