Pseudotargeted peptidomics approach for identifying species-specific biomarkers in fermented Cordyceps products: A...

Bailing Capsule, a fermented Cordyceps product used as an adjunctive treatment for chronic kidney disease and chronic bronchitis, is a crucial substitute for wild Ophiocordyceps. However, its current quality control relies on non-specific small-molecule markers, making it hard to distinguish from other analogous products. In this study, a pseudotargeted peptidomics strategy was developed to discover characteristic peptides for differentiation. Firstly, digested peptides were obtained by trypsin digestion (37 °C, 24 h, enzyme/protein ratio 1: 50) and profiled by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS). Subsequently, a multiple reaction monitoring (MRM) transition list was generated from the Q-TOF data using a "MRM-Ion Pair Finder" tool. These transitions were validated by ultra-high performance liquid chromatography triple-quadrupole mass spectrometry (UPLC-QQQ-MS) with 48 batches of samples from Bailing Capsule and four other analogous products. As a result, a total of 150 characteristic transitions specific to Bailing Capsule were identified, of which 76 peptide sequences were elucidated, and 12 peptide biomarkers were confirmed using synthetic standards. This pseudotargeted approach exhibits broad coverage and good sensitivity. Without requiring prior knowledge of peptide sequence, the transition-driven strategy reduces redundancy and enables focused, transition-based peptide differentiation. Compared to conventional small-molecule biomarkers, this peptide-based approach offers species-specific identification. This study demonstrates that a pseudotargeted peptidomics strategy may aid in the quality control of fermented Cordyceps products.