Proteomic identification of marker proteins and its application to authenticate Ophiocordyceps sinensis

Ophiocordyceps sinensis (O. sinensis) is a highly valuable fungus because of its nutritious and medicinal properties. The objective of this study was to identify protein markers using a proteomics approach followed by the development of an immunoassay to authenticate O. sinensis. Four authentic O. sinensis samples collected from four production regions and four counterfeit samples were examined individually. Overall 22 characteristic proteins of O. sinensis were identified by two-dimensional electrophoresis (2-DE) coupled with the matrix-assisted laser desorption/ionization-time-of-light mass spectrometry (MALDI-TOF/MS). Three authentic O. sinensis samples and three counterfeit samples were examined by the couple of alkaline native gradient PAGE (AN-PAGE) and electrospray ionization quadrupole-time-of-light mass spectrometry (ESI-Q-TOF/MS). One distinctive protein was identified to be cyanate hydratase, which was also one of the 22 distinctively characteristic proteins of O. sinensis and termed as IP4 in 2-D gel. Due to the abundance and high specificity of IP4, it was isolated and purified. Its purity was evaluated by high performance liquid chromatography (HPLC) and identified by ESI-Q-TOF/MS. Then the purified IP4 was used to produce polyclonal antibodies in BALB/c mice. The specificity of the anti-IP4 antibody was evaluated by an association of double immunodiffusion (DID) and indirect ELISA assay. Then an indirect enzyme linked immunosorbent assay (ELISA) was preliminarily developed to authenticate O. sinensis by detecting IP4. To evaluate the feasibility and accuracy of this method, three authentic O. sinensis samples and three counterfeits were analyzed. The P/N ratios (dividing the sample OD_450nm by the OD_450nm of negative controls) of three authentic O. sinensis samples were above 8, while, those of three counterfeits were lower than 1. These results indicated that the established ELISA assay based on proteomic protocols detection of protein markers might have a great potential in the authentication and also quality assessment of O.sinensis in those commercial products.