Jiao Fenglong, Gao Fangyuan, Liu Yuanyuan, Fan Zhiya, Xiang Xiaochao, Xia Chaoshuang, Lv Yayao, Xie Yuping, Bai Haihong, Zhang Wanjun, Qin Weijie, Qian Xiaohong
National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing, 102206, China.
Phase I Clinical Trial Center, Capital Medical University Affiliated Beijing Shijitan Hospital University, Beijing, 100038, China.
Talanta. 2021 Feb 1;223(Pt 2):121776. doi: 10.1016/j.talanta.2020.121776. Epub 2020 Oct 15.
Small extracellular vesicles (SEVs), are cell-derived, membrane-enclosed nanometer-sized vesicles that play vital roles in many biological processes. Recent years, more and more evidences proved that small EVs have close relationship with many diseases such as cancers and Alzheimer's disease. The use of phosphoproteins in SEVs as potential biomarkers is a promising new choice for early diagnosis and prognosis of cancer. However, current techniques for SEVs isolation still facing many challenges, such as highly instrument dependent, time consuming and insufficient purity. Furthermore, complex enrichment procedures and low microgram amounts of proteins available from clinical sources largely limit the throughput and the coveage depth of SEVs phosphoproteome mapping. Here, we synthesized Ti-modified magnetic graphene-oxide composites (GFST) and developed a "one-material" strategy for facile and efficient phosphoproteome enrichment and identification in SEVs from human serum. By taking advantage of chelation and electrostatic interactions between metal ions and phosphate groups, GFST shows excellent performance in both SEVs isolation and phosphopeptide enrichment. Close to 85% recovery is achieved within a few minutes by simple incubation with GFST and magnetic separation. Proteome profiling of the isolated serum SEVs without phosphopeptide enrichment results in 515 proteins, which is approximately one-fold more than those otained by ultracentrifugation or coprecipitation kits. Further application of GFST in one-material-based enrichment led to identification of 859 phosphosites in 530 phosphoproteins. Kinase-substrate correlation analysis reveals enriched substrates of CAMK in serum SEVs phosphoproteome. Therefore, we expect that the low instrument dependency and the limited sample requirement of this new strategy may facilitate clinical investigations in SEV-based transportation of abnormal kinases and substrates for drug target discovery and cancer monitoring.
小细胞外囊泡（SEVs）是细胞来源的、膜包裹的纳米级囊泡，在许多生物学过程中发挥着重要作用。近年来，越来越多的证据表明，小细胞外囊泡与许多疾病密切相关，如癌症和阿尔茨海默病。将SEVs中的磷酸化蛋白质用作潜在生物标志物，是癌症早期诊断和预后的一个有前景的新选择。然而，目前用于SEVs分离的技术仍面临许多挑战，如高度依赖仪器、耗时且纯度不足。此外，复杂的富集程序以及临床来源中可获得的蛋白质微克量低，在很大程度上限制了SEVs磷酸化蛋白质组图谱的通量和覆盖深度。在此，我们合成了钛修饰的磁性氧化石墨烯复合材料（GFST），并开发了一种“单一材料”策略，用于从人血清中简便、高效地富集和鉴定SEVs中的磷酸化蛋白质组。通过利用金属离子与磷酸基团之间的螯合和静电相互作用，GFST在SEVs分离和磷酸肽富集方面均表现出优异性能。通过与GFST简单孵育并进行磁分离，在几分钟内即可实现近85%的回收率。对未进行磷酸肽富集的分离血清SEVs进行蛋白质组分析，得到515种蛋白质，这比通过超速离心或共沉淀试剂盒获得的蛋白质数量多约一倍。GFST在基于单一材料的富集中的进一步应用，导致在530种磷酸化蛋白质中鉴定出859个磷酸化位点。激酶-底物相关性分析揭示了血清SEVs磷酸化蛋白质组中钙调蛋白依赖性蛋白激酶（CAMK）的富集底物。因此，我们期望这种新策略对仪器的低依赖性和对样品的有限需求，可能有助于基于SEV的异常激酶和底物转运的临床研究，以用于药物靶点发现和癌症监测。
