脑脊液外泌体亚群的分离及其蛋白质组学分析  

Isolation and proteomics analysis of cerebrospinal fluid exosome subtypes

作　　者：陈晓菲 刘威 张文镓 李言鹏 王志华 高明霞[1] 张祥民[1] CHEN Xiaofei;LIU Wei;ZHANG Wenjia;LI Yanpeng;WANG Zhihua;GAO Mingxia;ZHANG Xiangmin(Department of Chemistry,Fudan University,Shanghai 200438,China;Shanghai Pudong Hospital,Shanghai 201399,China)

机构地区：[1]复旦大学化学系,上海200438 [2]上海市浦东医院,上海201399

出　　处：《色谱》2025年第5期518-528,共11页Chinese Journal of Chromatography

基　　金：国家自然科学基金(22274027);上海市浦东新区卫生计生科研项目(PW2021A-70).

摘　　要：外泌体是一类尺寸为30~200 nm、携带有蛋白质等生物活性物质的细胞外小囊泡,能够分为具有不同功能的亚群,对外泌体异质性的了解有助于研究其在生理和病理中的作用机制。脑脊液来源的外泌体作为疾病的生物标志物具有巨大潜力,但是相关蛋白质组学的研究报道较少,且缺乏对脑脊液外泌体亚群的研究。本研究发展了串联体积排阻色谱法用于分离脑脊液外泌体亚群,并结合纳升级液相色谱-捕集离子淌度飞行时间质谱联用技术(nanoLC-TIMS-TOF-MS),对创伤性脑损伤患者的外泌体亚群进行了蛋白质组学分析。结果表明,利用串联体积排阻色谱法分离得到4个不同尺寸的脑脊液外泌体亚群,不同亚群共鉴定到739个蛋白质,进一步的蛋白质组学分析结果表明外泌体亚群与细胞信号转导、凝血过程和免疫反应等生物过程密切相关。此外,研究结果揭示了脑脊液外泌体亚群的蛋白表达存在异质性,特别是大尺寸的亚群在不同样本间存在较大的差异。综上,本研究利用开发的串联体积排阻色谱方法分离得到脑脊液外泌体亚群,并从蛋白质组学的角度丰富了对创伤性脑损伤患者脑脊液外泌体的认知。Exosomes are small extracellular vesicles 30-200 nm in diameter that contain many bioactive macromolecules,including proteins,lipids,and nucleic acids;consequently,they play important roles in many physiological and pathological processes and are classified into various property-dependent subtypes.Research into exosome heterogeneity helps broaden our understanding of the physiological and pathological mechanisms associated with exosomes.Exosomes exist in many human biological fluids,with those derived from cerebrospinal fluid(CSF)regarded as potential disease biomarkers.Despite this,few studies have focused on their proteomics,and little research into CSF-derived exosome subtypes has been reported.Traumatic brain injury(TBI)is a major public health issue characterized by a large number of patients and complex pathological processes.While a comprehensive understanding of the pathophysiological processes that underpin TBI is essential for developing therapeutic interventions,proteomic studies into CSF-derived exosomes in patients with TBI are limited.Herein,we designed a tandem size-exclusion chromatography protocol for isolating and profiling the proteins of CSF-derived exosome subtypes from patients with TBI using nanoscale liquid chromatography and trapped-ion mobility spectrometry time-of-flight mass spectrometry(nanoLC-TIMS-TOF-MS).We first centrifuged the collected CSF to remove cells and cell debris,after which it was concentrated by ultrafiltration to increase the exosome concentration and remove small proteins and peptides.A mini-size exclusion chromatography(Mini-SEC)column was then used to separate the exosomes from large amounts of interfering proteins,after which high performance liquid-SEC(HPL-SEC)was used to further separate exosomes according to size.The entire extracellular-vesicle-subset separation and purification process takes approximately 1 h for a single CSF sample.Four differently sized exosome subtypes were successfully isolated and are referred to as S1,S2,S3,and S4 in order of descending

关 键 词：外泌体 外泌体亚群 蛋白质组学 脑脊液 创伤性脑损伤 

分 类 号：O658[理学—分析化学]