Identification of Pinelliae Rhizoma and its counterfeit species based on enzymatic signature peptides from toxic proteins

Pinelliae Rhizoma (PR), a toxic medication, with long history, is commonly used for eliminating phlegm. Due to the shortage of wild resources and the relative lacking of cultivation technology, it is often confused with its counterfeit species in the market, such as Typhonii Rhizoma (TR), Arisaematis Rhizoma (AR) and tubers of Typhonium flagelliforme (TF) and Pinellia pedatisecta (PP). It was aimed to screen signature enzymatic peptides from toxic proteins to identify PR and its four counterfeit species. A comparative proteogenomics strategy based on open-source transcriptome data was applied for screening signature peptides from toxic proteins, which were applied for species authentication of PR and its counterfeit species. Firstly, the open-source transcriptome data was used for constructing the annotated protein database, which was used for peptides identification. Secondly, the toxicity of different fractions of PR were evaluated by the rat peritoneal inflammation model. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to profile the main proteins bands of five species, whose sequences were identified based on the in-gel digestion experiment by using ultra-high-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry. Finally, the label-free proteomic analysis was performed to character the proteins and screen the signature peptides of five species, which were validated in commercially available products by dynamic multi reaction monitor (DMRM). The results in this study confirmed that protein was the main toxic components of PR. Both Pinellia ternata agglutinin (PTA) and trypsin inhibitor (TI) like proteins are the main proteins, which were characterized by proteomic analysis based on four annotated protein database. Meanwhile, seven signature peptides from toxic proteins were screened and validated with good repeatability and specificity in commercial products. Seven signature enzymatic peptides from toxic protein screened by the comparative proteogenomics strategy based on open-source transcriptome data achieved good identification ability of PR and its four counterfeit species.