使用邻近编码技术PBA分析单个外泌体上的表面蛋白质
前言
外泌体已涉及许多生物过程，并且它们可以作为重要的疾病标记物。外泌体上的表面蛋白质携带有关其来源组织的信息。由于外泌体的异质性，需要单独研究它们，但迄今为止这仍然是不切实际的。8月26日乌普萨拉大学生命科学实验室免疫学，遗传学和病理学团队在Nature Communications查看期刊详情上发表了“Profiling surface proteins on individual exosomes using a proximity barcoding assay”，文章展示了一种邻近编码技术分析法，用于使用抗体-DNA偶联物和下一代测序来分析各个外泌体的表面蛋白。首先使用人工链霉抗生物素蛋白-寡核苷酸复合物验证该方法，然后分析来自人体体液或细胞培养基的各个外泌体上的表面蛋白的可变组成。来自不同来源的外泌体的特征在于表面蛋白的特定组合及其丰度的存在，允许外泌体在混合样品中单独定量，以用作疾病中组织特异性参与的标志物。
研究思路
Figure 1：Design and workflow of PBA. a ，Preparation of PBA probes by chemical conjugation of antibodies and DNA oligonucleotides containing an 8-nt proteinTag and an 8-nt random sequence moleculeTag.b,Preparation of RCA products from circularized oligonucleotides comprising a 15-nt random sequence as complexTags, capable of encoding in excess of one billion unique complexTags. c，To profile surface proteins of exosomes by PBA, exosomes are first incubated with PBA probes, followed by capture of exosomes with bound PBA probes in microtiter wells via immobilized cholera toxin subunit B (CTB). Oligonucleotides on PBA probes brought together by binding the same exosome are next allowed to hybridize to a unique RCA product, followed by enzymatic extension, incorporating the complexTag present in the RCA product along with a standard sequence motif, later used for amplification. To prevent the DNA polymerase from extending across nearby monomers in the RCA products, blocking oligonucleotides are pre-hybridized to the RCA products. Successfully extended DNA molecules on PBA probes are amplified by PCR using the PCR primer1 and PCR primer2 for library preparation, while oligonucleotides on the antibodies or RCA products fail to be amplified by the PCR primer pairs used for amplification. The PCR product were subjected to DNA sequencing to record the numbers of molecules with specific tag combinations, thus revealing the identities of proteins on individual exosomes.
Figure 2：Validation of PBA using STV-biotin-oligonucleotide complexes. Tetrameric streptavidin molecules were incubated with four different biotinylated oligonucleotides, either separately or in a mixture of all four oligonucleotides. Streptavidin and biotinylated oligonucleotides were combined at molar ratios of 1:10 or 10:1. a, The numbers of observed complexes with different numbers of proteinTags and moleculeTags are summarized in pie charts. b, When the oligonucleotides were in excess, the numbers of complexes from separate or mixed oligonucleotides incubation with 2, 3, or 4 moleculeTags were grouped according to the number of proteinTags per complex.
Figure 3：Validation of PBA on exosomes using antibodies to CD9 and CD63. a Separate incubation: exosomes isolated from the K562 cell line were incubated with two distinct sets of PBA probes separately before they were pooled for PBA. b Mixed incubation: exosomes were incubated with four different PBA probes before PBA was performed.
Figure 4：Bulk protein counts and visualization of individual exosomes. a, b Exosomes from 18 different samples were analyzed for the presence of 38 proteins (40 proteinTags) by PBA. a Heatmaps representing the total amounts of the proteins by log(moleculeTag+1) found on exosomes from different sources. b Individual exosomes with one identified protein type, two protein types, and three or more protein types were visualized by t-SNE according to their protein compositions, with color and shape of the symbols representing the source of each exosome. Cells from two regions dominated by exosomes from the cell lines AGS and BPH-1 are highlighted in circles with dotted contours.
Figure 5：Quantification of exosomes from different sources according to their surface protein combinations, as revealed by PBA. a, Protein combinations selective for either K562 exosomes (orange) or prostasomes (blue), compared to exosomes from serum (gray), were sorted based on the number of exosomes with specific combinations. Here, the top 50 for each exosome are displayed. b, c, Serial dilution of K562 exosomes or prostasomes spiked in serum exosomes were quantified using K562- or prostasomes-selective combinations, respectively. The exosomes identified with either K562- or prostasome-selective combinations are indicated by orange and blue filled circles in the tSNE plots in b, and with orange and blue bars, respectively, in the bar plots in c. The sizes of the colored circles in (b) are proportional to the number of exosomes with the same protein combination.
结论
生物样品中不同种类的外泌体的表示可以提供有价值的诊断信息，但是目前的外泌体分析方法不容易获得该信息。在生物标志物发现中，可能对疾病具有诊断价值的外泌体物种可能以低丰度存在于样品中的其他外泌体群体中，从而模糊了任何诊断价值。文章使用DNA测序解码抗体与蛋白质特异性DNA标签的组合，结合单个外泌体，通过DNA延伸与大量DNA束的单个成员-RCA产品相关联-多个拷贝特定的随机DNA标签-复合标签。已经使用该测定来鉴定单个外泌体的蛋白质组成。PBA可以识别和量化来自单个外泌体或任何其他蛋白质簇的给定组的大量蛋白质组合。
PBA在微量滴定孔中进行，无需在纳米孔或液滴中进行区室化或使用特殊设备，通过使用由RCA产生的大量亚微米大小的DNA束，其大小与外泌体相似。基于phi29DNA聚合酶的已知合成速率，在复制10分钟后产生代表具有~200个补体的100-nt DNA环的DNA多联体的RCA产物。因此，这些RCA产品中的每一种都含有约200个拷贝的独特复合物标签，其可以与蛋白质标签组合以鉴定存在于个体外泌体上的蛋白质。PBA提供了巨大的多路复用能力，因为目前有40种探针，专门针对38种不同的蛋白质，可以轻松扩展到更大的数量.文章证明PBA可用于通过其可变表面蛋白质组合物区分外泌体，从而能够分析异质样品中不同的外泌体群体。
PBA的检测限取决于给定蛋白质组合的特异性，测序的覆盖度和深度，以及抗体的亲和力和选择性。为了增加检测来自给定组织来源的外泌体水平升高的可能性，靶向组织特异性蛋白质并将蛋白质组扩展到目前的38种蛋白质之外可能有助于进一步降低检测限。除了外泌体或其他细胞外囊泡表面上的那些之外，本文描述的技术还应适合于绘制其他更高级的蛋白质复合物。
总之，文章开发了一种分析单个外泌体表面蛋白的技术，采用抗体-DNA结合物和在大量独特RCA产物中重复的独特标签序列的组合。通过比较来自不同来源的外泌体的谱，可以找到特定的表面蛋白质组合，并用于鉴定和定量可能从健康和疾病中的特定组织释放到血液的非常大量的外泌体。
全文链接:
https://pubmed.ncbi.nlm.nih.gov/31451692/
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