Summary: | <i>Dipsacus asperoides</i> is a kind of Chinese herbal medicine with beneficial health properties. To date, the quality of <i>D. asperoides</i> from different habitats has shown significant differences. However, the molecular differences in <i>D. asperoides</i> from different habitats are still unknown. The aim of this study was to investigate the differences in protein levels of <i>D. asperoides</i> from different habitats. Isobaric tags for relative and absolute quantification (iTRAQ) and 2DLC/MS/MS were used to detect statistically significant changes in <i>D. asperoides</i> from different habitats. Through proteomic analysis, a total of 2149 proteins were identified, of which 42 important differentially expressed proteins were screened. Through in-depth analysis of differential proteins, the protein metabolism energy and carbohydrate metabolism of <i>D. asperoides</i> from Hubei Province were strong, but their antioxidant capacity was weak. We found that three proteins, UTP-glucose-1-phosphate uridylyltransferase, allene oxide cyclase, and isopentyl diphosphate isomerase 2, may be the key proteins involved in dipsacus saponin VI synthesis. Eight proteins were found in <i>D. asperoides</i> in response to environmental stress from different habitats. Quantitative real-time PCR analysis confirmed the accuracy and authenticity of the proteomic analysis. The results of this study may provide the basic information for exploring the cause of differences in secondary metabolites in different habitats of <i>D. asperoides</i> and the protein mechanism governing differences in quality.
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