中药蛲虫，作为传统的中药，具有促进血液循环和祛瘀血的功效，主要用于心血管和脑血管疾病的临床治疗。发现死亡和干燥的药用蛲虫中的活性蛋白或肽是一项具有重要挑战性的任务。本研究的目的是提供基本的蛋白质组学简介，并帮助进一步发现药用蛲虫中的活性蛋白质和质量控制措施，同时为动物药物研究提供洞察力。 从药材市场采集了17批次干燥的药用蛲虫，涵盖了三个物种，通过DNA条形码鉴定确保了其真实性。然后，通过无标记蛋白质组学技术对不同物种的蛲虫进行了蛋白质组学分析，以揭示显著不同的蛋白质。基于生物途径分析筛选出特征性肽段，并使用稳定同位素标记的平行反应监测方法进行绝对定量。 这17批次的蛲虫材料分别为Whitmania pigra Whitman (WP)，Whitmania laevis Whitman (WL)和Poecilobdella manillensis Lesson (PM)。共鉴定了1,035种蛋白质（WP 452种，WL 425种，PM 158种）。其中，90种重叠蛋白主要集中在多样化的代谢途径中，并主要定位于细胞质和线粒体内膜，主要与ATP结合、催化活性和结构分子活性等相关。总共鉴定出51种特异性表达的蛋白质（WP 21种，WL 23种，PM 7种），与多个关键信号通路相关，包括Rap1、cGMP-PKG、PI3K-Akt、Wnt和HIF-1等，与心血管疾病、糖尿病、癌症甚至多种神经退行性疾病的治疗相关。选择具有潜在生物活性的三种蛋白质（神经血红蛋白、血蛤素和Eglin C）作为质量指标，并基于特征性肽段进行定量。 本研究描述了三种蛲虫的蛋白质组学简介，探究了药用蛲虫的潜在活性蛋白质，并有望为疾病治疗的潜在分子机制提供帮助。本研究中的基于蛋白质组学的方法不仅有助于发现具有潜在生物活性的蛋白质，还有助于动物药物的生物活性相关质量控制。 Copyright © 2023. Published by Elsevier B.V.

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.Copyright © 2023. Published by Elsevier B.V.