尿液细胞外囊泡的高效亲和纯化捕获与蛋白质组学分析  

Efficient capture and proteomics analysis of urinary extracellular vesicles by affinity purification

作　　者：张贵元 詹震 陶纬国 张昊 ZHANG Guiyuan;ZHAN Zhen;TAO Weiguo;ZHANG Hao(School of Biological Science&Medical Engineering,Southeast University,Nanjing 210096,China;EVLiXiR Biotech,Nanjing 210032,China;Bell Mountain Molecular MedTech Institute,Nanjing 210032,China;Department of Biochemistry,Purdue University,West Lafayette,IN 47907,America)

机构地区：[1]东南大学生物科学与医学工程学院,江苏南京210096 [2]逸微健华生物科技有限公司,江苏南京210032 [3]南京紫金山分子医学技术研究院,江苏南京210032 [4]普渡大学生物化学系,印第安纳州西拉法叶47907

出　　处：《色谱》2025年第5期508-517,共10页Chinese Journal of Chromatography

基　　金：国家重点研发计划(2017YFA0700404).

摘　　要：目前,液体活检因其创伤小、易获取且操作简便的优点,已成为癌症诊断中传统组织活检的替代方法。尿液来源的细胞外囊泡(extracellular vesicles,EVs)已被确定为癌症生物标志物的重要来源。EVs是由细胞分泌的磷脂双分子层囊泡,内含蛋白质、DNA及RNA等成分,且由于双层磷脂膜的保护,EVs内的蛋白质可免受体液中酶的降解。然而,在蛋白质组学分析中,低产率使得从生物体液中分离EVs仍是一个挑战。在本研究中,我们采用亲和磁珠EVlent对尿液中的EVs进行富集,EVlent通过识别EVs表面的特异性蛋白质实现高选择性富集。首先使用蛋白免疫印迹、纳米粒子追踪分析和透射电子显微镜对尿液中的EVs进行综合表征,结果表明,EVlent亲和磁珠成功的从尿液中分离出了EVs,且富集效果优于传统的超速离心方法。接下来,将此方法应用于15例健康志愿者和15例前列腺癌患者的尿液EVs蛋白质组学分析,并筛选潜在的肿瘤标志物。结果显示,健康对照组平均鉴定出2039种蛋白质和14490条肽段,前列腺癌患者中分别鉴定出1982种蛋白质和13100条肽段。进一步分析发现,91种蛋白质属于Vesiclepedia数据库中最常见的100种EV蛋白质。与健康对照组相比,前列腺癌患者尿液EVs中88种蛋白质表达上调,90种蛋白质下调。KEGG分析揭示4种蛋白质(尿激酶型纤溶酶原激活剂(PLAU)、血小板衍生生长因子A(PDGFA)、基质金属蛋白酶3(MMP3)和神经母细胞瘤RAS病毒癌基因同源物(NRAS))在前列腺癌通路中富集,这些蛋白质未来有希望作为前列腺癌的潜在生物标志物,可以为前列腺癌的早期诊断及预后提供重要依据。Liquid biopsy is a promising alternative to traditional tissue biopsies for diagnosing cancer because it offers advantages such as minimal invasiveness,accessibility,and ease of operation.Extracellular vesicles(EVs)are lipid bilayer vesicles that contain proteins,DNA,and RNA and are secreted by cells.Indeed,urinary EVs are important sources of cancer biomarkers.The lipid bilayer protects EV proteins from degradation by enzymes present in bodily fluids.Prostate cancer(PCa)is among the most prevalent malignancies in developed countries and is the second-leading cause of cancer-related mortality in men.Current screening methods commonly used to initially evaluate patients with suspected PCa include serum prostate-specific antigen(PSA)testing and digital rectal examination(DRE),with magnetic resonance imaging(MRI)and transrectal ultrasound often recommended for further assessment.However,both PSA testing and DRE have limited specificities,which results in a substantial number of unnecessary prostate biopsies.Consequently,additional reliable biomarkers need to be urgently discovered for rapidly diagnosing PCa more accurately.Prostate-derived secretions,including those associated with malignancies,are detectable in urine owing to the anatomical proximity of the prostate to the urethra;hence urine is a promising liquid-biopsy medium for discovering PCa biomarkers,which is a topic that has been the focus of extensive research efforts in recent years.However,isolating EVs from biofluids in sufficient yields for proteomics analysis remains challenging.In this study,functional magnetic beads EVlent(extracellular vesicles isoLated efficiently,naturally,and totally)with high-affinity capabilities were developed for selectively enriching EVs from biological fluids.The surfaces of the beads were modified with three antibodies that target CD9,CD63,and CD81,which enables the specific recognition of EV surface proteins.The isolation performance of EVlent was validated by comprehensively characterizing urinary EVs using Western blo

关 键 词：细胞外囊泡 尿液 蛋白质组学 前列腺癌 亲和纯化 

分 类 号：O658[理学—分析化学]