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Leech, a classical traditional Chinese medicine for promoting blood circulation and removing blood stasis, is mainly used in the clinical treatment of cardiovascular and cerebrovascular diseases. The discovery of activity proteins or peptides in the dead and dried medicinal leech is an important task with great challenges. The aim of this study was to provide a basic proteome profile and help further discover active proteins and quality control for medicinal leeches, which would also provide insight into the research of animal medicines. Seventeen batches of dried medicinal leeches covering three species were collected from medicinal markets, which were authenticated by DNA barcoding. Then the proteome of different species leeches was profiled to reveal the significantly different proteins using label-free proteomics. The characteristic peptides were screened out based on biological pathways analysis, which were further absolutely quantified using the developed stable isotope-labeled based parallel reaction monitoring method. Seventeen batches of leech materials were Whitmania pigra Whitman (WP), Whitmania laevis Whitman (WL) and Poecilobdella manillensis Lesson (PM), respectively. A total of 1,035 proteins (452 in WP, 425 in WL and 158 in PM) were identified. Among them, 90 overlapping proteins were mainly concentrated in diverse metabolic pathways and primarily localized in the cytoplasm and mitochondrial inner membrane, which mainly related to ATP binding, catalytic activity and structural molecular activity. In total of 51 uniquely expressed proteins (21 in WP, 23 in WL and 7 in PM), associated with multiple key signaling pathways, including Rap1, cGMP-PKG, PI3K-Akt, Wnt and HIF-1, etc., relevant to treating cardiovascular diseases, diabetes, cancer and even a variety of neurodegenerative diseases. Three proteins with potential bioactivities, including Neurohemerythrin, Hirudin and Eglin C, were selected as the quality makers and then quantified based on the characteristic peptides. This work profiled the proteome of three species of leeches, and addressed potential active proteins of the medicinal leech, which would help to provide the potential molecular mechanisms involved in disease treatment. The proteomics-based approach developed in this work is not only useful for the discovery of proteins with potential bioactivities but also helpful for the bioactivity relevant quality control of animal medicines. [Display omitted] • 17 batches of commercial leeches were identified using DNA barcoding based on COI gene. • A total of 1035 proteins were identified from 3 species leeches. • 90 co-existed proteins and 51 uniquely expressed proteins were characterized. • A characteristic peptides-based parallel reaction monitoring quantitative approach was developed. • Proteome profiling helps to discover active proteins of medicinal leeches. [ABSTRACT FROM AUTHOR]