Development of a targeted mass spectrometry method for porcine pancreas marker peptides detection in protein hydrolysates.

Protein hydrolysates are widely used in health foods, but undisclosed enzymatic sources, particularly porcine pancreas, raising concerns regarding food safety, allergenicity, and dietary restrictions. To address this issue, this study comprehensively characterized the porcine pancreas proteome, identifying 418 proteins and establishing 50 °C autolysis to ensure consistent peptide profiles. Three homologous highly specific α-amylase-derived marker peptides, VTNPSRPW, VVTNPSRPW, and IVVTNPSRPW, were selected and validated, and a targeted LC-MS/MS assay was established with porcine pancreas detection limits of 0.5 %, 1 %, and 5 % (w/w), respectively. Application of this method to commercial products revealed the presence of porcine peptides in samples labeled as "animal-free" which was further proved by on-site investigation. This study provides a robust and practical approach for verifying the species origin of enzymatic preparations in protein hydrolysates, enhancing traceability, food safety, and regulatory compliance. To our knowledge, it represents the first systematic investigation into enzyme source authenticity in such products.