磷脂酰丝氨酸分子印迹聚合物对血浆外泌体的富集与蛋白质组学分析  

Enriching plasma exosomes for proteomic analysis using a phosphatidylserine-imprinted polymer

作　　者：程显惠 于文静 王冬雪 姜丽艳[1] 胡良海[1] CHENG Xianhui;YU Wenjing;WANG Dongxue;JIANG Liyan;HU Lianghai(School of Life Sciences,Jilin University,Changchun 130023,China;Theπ-HuB Project Infrastructure,International Academy of Phronesis Medicine,Guangzhou 510000,China)

机构地区：[1]吉林大学生命科学学院,吉林长春130023 [2]慧眼大科学设施,智慧医学国际研究院,广东广州510000

出　　处：《色谱》2025年第5期539-546,共8页Chinese Journal of Chromatography

基　　金：国家自然科学基金(22374056);人体蛋白质组导航计划(P0020).

摘　　要：外泌体是肿瘤标志物的重要来源,血浆作为最常用的临床体液之一,其组成较为复杂且存在大量高丰度蛋白质的干扰,如何有效地从血浆中分离外泌体是临床研究的重要挑战之一。本研究将磷脂酰丝氨酸分子印迹聚合物(PS-MIP)用于血浆外泌体的富集,PS-MIP能够特异性识别外泌体质膜上的磷脂酰丝氨酸,从而实现外泌体的高选择性富集。该方法被用于3例健康志愿者和3例胰腺癌患者的血浆蛋白质组学分析和潜在肿瘤标志物的筛选,在健康对照组中的血浆外泌体中鉴定到了1052种蛋白质和4545条肽段,胰腺癌患者血浆外泌体中鉴定到了972种蛋白质和4096条肽段。将蛋白质组学鉴定到的外泌体蛋白质与包含所有细胞外囊泡分子信息的Vesiclepedia数据库进行比较,结果表明84%的PS-MIP富集的血浆外泌体蛋白质存在于该数据库;与只包含外泌体分子信息的ExoCarta数据库进行比较,发现PS-MIP法鉴定出了ExoCarta数据库中Top 100外泌体蛋白质中的77种。与健康对照组相比,胰腺癌患者血浆外泌体中表达量上调的蛋白质有11个,下调的蛋白质有24个。蛋白相互作用网络(PPI)分析显示,相关性较高的前3个蛋白质是补体因子D(CFD)、补体C3(C3)和血管性血友病因子(VWF),在胰腺癌患者外泌体的蛋白质组学表达上调的蛋白质中,外泌体蛋白样糖基转移酶2(EXTL2)、α-2-巨球蛋白样1(A2ML1)和人帕金森病蛋白7(PARK7)的差异最为显著,这些蛋白质可能是胰腺癌诊断和预后评估的潜在生物标志物,为胰腺癌的早期诊断和预后提供了重要的科学依据。Exosomes are 40-160 nm vesicular nano-bodies secreted by most cells that carry large amounts of biologically active substances originating from the parent cell.Proteins in exosomes are protected by phospholipid bilayer membranes that protect them from degradation by enzymes within body fluids.Along with nucleic acid,proteins and metabolites,exosomes are biomolecules that are considered to be among the most important for discovering tumor markers.Plasma is among the most commonly used body fluids in clinical settings;it is highly complex and contains many proteins and metabolites that interfere with exosome isolation.Consequently,the development of methods for effectively isolating exosomes is a key challenge prior to their use in clinical research.In this study,we used a phosphatidylserine molecularly imprinted polymer(PS-MIP)to enrich plasma exosomes.Subsequent immunoblotting analyses for the CD9,TSG101,and CD81 exosome marker proteins showed that signals can be detected using only 5μL of plasma,thereby demonstrating the efficiency and specificity of the enrichment protocol.Transmission electron microscope(TEM)and nanoparticle tracking analysis(NTA)data revealed that the enriched vesicles are 30-100 nm in size with elliptical or cup-shaped structures,consistent with the morphology and particle-size-distribution characteristics of the exosomes,suggesting that PS-MIP is capable of successfully isolating exosomes.Nanoflow cytometry revealed that 75.4%of the multi-angle laser scattering(MALS)signal is derived from the PS-MIP-enriched exosomes,which indicates that these enriched exosomes are highly pure and free of interference from impurities,such as aggregated protein particles that are similar in size to the exosomes themselves.This method was used to analyze the proteomes and potential exosomal protein markers of clinical plasma samples from three pancreatic-cancer patients and three healthy volunteers.A total of 1052 proteins and 4545 peptides were identified in the plasma exosomes of healthy volunteers,with a

关 键 词：液相色谱-串联质谱 外泌体 血浆 蛋白质组学 胰腺癌 分子印迹 磷脂 

分 类 号：O658[理学—分析化学]