A novel strategy to identify the species-specific peptide biomarkers in Pheretima aspergillum (E. Perrier) based on enzymatic digestion followed by LC-MS/MS methods.

Guang Dilong [P. aspergillum (E. Perrier)], is an animal-derived traditional Chinese medicine made from the dried body of Pheretima aspergillum (E. Perrier) (TCM). Due to its widely application and high medical values, preparations of P. aspergillum (E. Perrier) may be adulterated by four other species, including three crucial Pheretima species [P. vulgaris (Chen), P. pectinifera (Mkhaeken), and P. guillemi (Michaelsen)] and one considerable adulteration [Metaphire magna (Chen)]. This study developed a novel and effective strategy for analyzing and authenticating Guang Dilong based on enzymatic digestion of protein. The nanoLC-MS/MS technique used to evaluate complete peptidomics profiles of trypsin-digested samples, resulting in the identification of species-specific peptide biomarkers in P. aspergillum (E. Perrier). The significance of different samples and peptides in the target species set was then investigated using mathematical set theory. Consequently, seven peptides were chosen as prospective biomarkers. Finally, five specific peptide biomarkers for differentiating Guang Dilong with other species were confirmed and validated using UFLC-MS/MS and MRM mode. The suggested technique may also be beneficial in evaluating the quality of other animal-derived goods for safety issues in order to avoid misidentification.