High-throughput Screening of Potential Allergens from Complex Proteins of Large Yellow Croaker (Larimichthys crocea) by Mass Spectrometry

Abstract

Abstract The allergenicity of large yellow croaker (Larimichthys crocea) proteins has been widely reported to cause many serious food safety problems. In this study, the protein sample extracted from Larimichthys crocea was digested and analyzed by reversed phase liquid chromatography coupled with tandem mass spectrometry (RPLC-MS/MS) for globally proteomic analysis, and the Larimichthys crocea proteins were digested with simulated gastric fluid (SGF) at different time intervals and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The digestion-resistant proteins were further done with in-gel digestion and analyzed by RPLC-MS/MS. Then, sequence homology analysis between identified proteins and known allergens was performed. Results showed that 33 kinds of distinct proteins were feasibly identified from Larimichthys crocea by RPLC-MS/MS and the protein band (approximately 44.0 kDa) presented in SDS-PAGE was considered as protein-resistant to SGF digestion. Two potential allergens, β-enolase and tropomyosin α−1 chain, were found from proteins resistant to SGF digestion by sequence homology analysis. It was observed that most of these SGF digestion-resistant proteins were potential allergens with high sequences similarity to the known protein allergens from other species. Therefore, this strategy provides an efficient alternative for potential allergens screening with high-throughput, which exhibits great potential application in fish protein allergens related food safety control.