Peptidomics-based identification of endogenous free peptides from Ophiocordyceps sinensis and functional validation of immunomodulatory peptide WALGT

Ophiocordyceps sinensis (formerly known as Cordyceps sinensis), commonly referred to as “dongchongxiacao” in Chinese, is a valuable traditional Chinese medicine whose bioactive small molecules form the essential material basis for its therapeutic effects. Nonetheless, endogenous free peptides, as a class of potentially important active constituents, have remained relatively underexplored due to the high cost of Ophiocordyceps sinensis. In this study, peptidomics technology combined with the confidence scoring mechanism of MaxQuant analytic software (Andromeda score) was employed. Peptides for which each amino acid residue corresponds to a unique fragment ion signal in the tandem mass spectrometry (MS/MS) spectrum were designated as high-confidence identified peptides. High-confidence peptide samples were synthesized using solid-phase technology and subsequently verified using reverse comparative analysis of MS/MS fragmentation spectra under identical mass spectrometric conditions. This approach further ensured the accuracy of sequence analysis for endogenous free peptides in Ophiocordyceps sinensis. Our method requires merely milligram quantities of Ophiocordyceps sinensis samples for the detection of endogenous free peptides, which is a notable methodological improvement over the traditional activity-guided stepwise separation methods with high sample consumption used in previous peptide studies. Different from previous reports focusing on artificially enzymatically hydrolyzed peptides/peptide-like compounds, we identified native endogenous free peptides independent of proteolysis in Ophiocordyceps sinensis, and using RAW 264.7 cell culture, the peptide WALGT was confirmed to exert immunomodulatory effects via promoting macrophage proliferation. The proliferative effect was additionally confirmed using scratch test, colony formation assay, and proliferation factor analysis. Using a mouse immunosuppression model, the findings indicated that WALGT treatment markedly reinstated monocyte/macrophage and white blood cell (WBC) counts and the expression levels of immunological factors (IgG, IgM) in CTX-exposed mice relative to the model group, confirming the ability to enhance the immunological function of immunosuppressed mice. This study aims to provide a novel research strategy for analyzing endogenous free bioactive peptides using only milligram-level samples.