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Prunella vulgaris L., better known as 'self-heal', has been extensively used in the traditional system of medicines. To reveal the regulatory mechanism of its development, TMT-based quantitative proteome analysis was performed in the Prunella vulgaris L. spica before and during ripening (Group A and Group B, respectively). This analysis resulted in the identification of 7655 proteins, of which 1910 showed differential abundance between the two groups. Pronounced changes in the proteomic profile included the following: 1) Stress-responsive proteins involved in protecting cells and promoting fruit ripening and seed development were highly abundant during ripening. 2) The degradation of chlorophyll, inhibition of chlorophyll biosynthesis and increased abundance of transketolase occurred simultaneously in the spica of Prunella vulgaris L., resulting in the spica changing color from green to brownish red. 3) The abundance of protein species related to phenylpropanoid biosynthesis mainly increased during ripening, while flavonoid and terpenoid backbone biosynthesis mostly occurred before ripening. This study establishes a link between protein profiles and mature phenotypes, which will help to improve our understanding of the molecular mechanisms involved in the maturation of Prunella vulgaris L. at the proteome level and reveal the scientific connotation for the best time to harvest Prunella vulgaris L. This work provides a scientific basis for the production of high-quality medicinal Prunella vulgaris L., as well as a typical demonstration of molecular research used for the harvest period of traditional Chinese medicine. This work provided a comprehensive overview on the functional protein profile changes of Prunella vulgaris L. spica at different growing stages, as well as the scientific rationale of Prunella vulgaris L. harvested in summer after brownish red, thus laid an intriguing stepping stone for elucidating the molecular mechanisms of quality development. Unlabelled Image • Prunella vulgaris L. spica was sampled at five developmental stages (from heading to physiological maturity). • LC-MS matched 7655 proteins, of which 6405 can be quantified and 1910 proteins showed differences in abundance. • High abundance of stress-responsive protein species involved in fruit maturity and seed development during spica ripening. • The up-representation of transketolase, the degradation and inhibition synthesis of chlorophyll cause the spica changing color. • Increased levels of protein related to phenylpropanoid biosynthesis were observed during spica ripening [ABSTRACT FROM AUTHOR]