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Abstract：
Small extracellular vesicles (sEVs) are important mediators of intercellular communication by transferring of functional components (proteins, RNAs, and lipids) to recipient cells. Some PTMs, including phosphorylation and N-glycosylation, have been reported to play important role in EV biology, such as biogenesis, protein sorting and uptake of sEVs. MS-based proteomic technology has been applied to identify proteins and PTM modifications in sEVs. Previous proteomic studies of sEVs from C2C12 myoblasts, an important skeletal muscle cell line, focused on identification of proteins, but no PTM information on sEVs proteins is available.In this study, we systematically analyzed the proteome, phosphoproteome, and N-glycoproteome of sEVs from C2C12 myoblasts with LC-MS/MS. In-depth analyses of the three proteomic datasets revealed that the three proteomes identified different catalogues of proteins, and PTMomic analysis could expand the identification of cargos in sEVs. At the proteomic level, a high percentage of membrane proteins, especially tetraspanins, was identified. The sEVs-derived phosphoproteome had a remarkably high level of tyrosine-phosphorylated sites. The tyrosine-phosphorylated proteins might be involved with EPH-Ephrin signaling pathway. At the level of N-glycoproteomics, several glycoforms, such as complex N-linked glycans and sialic acids on glycans, were enriched in sEVs. Retrieving of the ligand-receptor interaction in sEVs revealed that extracellular matrix (ECM) and cell adhesion molecule (CAM) represented the most abundant ligand-receptor pairs in sEVs. Mapping the PTM information on the ligands and receptors revealed that N-glycosylation mainly occurred on ECM and CAM proteins, while phosphorylation occurred on different categories of receptors and ligands. A comprehensive PTM map of ECM-receptor interaction and their components is also provided.In summary, we conducted a comprehensive proteomic and PTMomic analysis of sEVs of C2C12 myoblasts. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analysis of sEVs might provide some insights about their specific uptake mechanism.
摘要：
小细胞外囊泡（sEV）是通过转移功能成分（蛋白质，RNA,和脂质）到受体细胞。一些PTM，包括磷酸化和N-糖基化，据报道，在EV生物学中起着重要作用，如生物发生，蛋白质分选和sEV的摄取。基于MS的蛋白质组学技术已用于鉴定sEV中的蛋白质和PTM修饰。以前对C2C12成肌细胞sEV的蛋白质组学研究，一种重要的骨骼肌细胞系，专注于蛋白质的鉴定，但没有关于sEV蛋白的PTM信息可用。在这项研究中,我们系统分析了蛋白质组，磷酸化蛋白质组，用LC-MS/MS和C2C12成肌细胞的sEV的N-糖蛋白组。对三个蛋白质组数据集的深入分析显示，这三个蛋白质组鉴定出蛋白质的不同目录，PTMomic分析可以扩展sEV中货物的识别。在蛋白质组层面,高比例的膜蛋白，尤其是四跨膜蛋白，已确定。sEV衍生的磷酸蛋白质组具有非常高水平的酪氨酸磷酸化位点。酪氨酸磷酸化蛋白可能与EPH-Ephrin信号通路有关。在N-糖蛋白质组学的水平，几种糖型，如复杂的N-连接聚糖和聚糖上的唾液酸，丰富了电动汽车。恢复sEV中的配体-受体相互作用表明，细胞外基质（ECM）和细胞粘附分子（CAM）代表了sEV中最丰富的配体-受体对。在配体和受体上绘制PTM信息显示N-糖基化主要发生在ECM和CAM蛋白上,而磷酸化发生在不同类别的受体和配体上。还提供了ECM-受体相互作用及其组分的全面PTM图谱。总之,我们对C2C12成肌细胞的sEVs进行了全面的蛋白质组学和PTMomic分析。整合蛋白质组学，磷酸化蛋白质组，和sEV的N-糖蛋白质组学分析可能为其特定的摄取机制提供一些见解。
