Multiplex protein profiling method for extracellular vesicle protein detection.
Multiplex protein profiling method for extracellular vesicle protein detection.
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DOI：
10.1038/s41598-021-92012-6
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发表时间：
2021-06-14
影响因子：
4.6
通讯作者：
Meckes DG Jr
中科院分区：
文献类型：
--
作者：
Sun L;Meckes DG Jr
细胞外囊泡（extracellular vesicles，EV）是由所有细胞分泌到生物液体中的小的纳米尺寸的膜囊。EV包裹来自其起源细胞的蛋白质、RNA和代谢产物，并在细胞间通讯事件中发挥重要作用。在过去的十年中，电动汽车已成为癌症诊断的新兴来源。EV研究及其作为诊断生物标志物的效用的挑战之一是传统蛋白质分析方法所需的EV量。在这里，我们提出了一种新的免疫PCR方法，该方法利用市售的TotalSeq抗体含有DNA缀合的寡核苷酸，使用实时qPCR鉴定固定化蛋白质分析。使用这种方法，我们证明了多种EV表面蛋白可以同时进行分析，具有高灵敏度和特异性。该方法也成功地应用于使用细胞和血清样品的类似方案。我们进一步描述了一种微尺寸排阻色谱法的发展，当与免疫PCR结合时，我们能够用10 μL的人血清检测EV表面蛋白。总体而言，这些结果表明，免疫PCR方法可以在单个试管中从小样品体积中快速检测多种EV标志物。
Extracellular vesicles (EVs) are small nanometer-sized membrane sacs secreted into biological fluids by all cells. EVs encapsulate proteins, RNAs and metabolites from its origin cell and play important roles in intercellular communication events. Over the past decade, EVs have become a new emerging source for cancer diagnostics. One of the challenges in the study of EVs and there utility as diagnostic biomarkers is the amount of EVs needed for traditional protein analysis methods. Here, we present a new immuno-PCR method that takes advantage of commercially available TotalSeq antibodies containing DNA conjugated oligos to identify immobilized protein analysts using real-time qPCR. Using this method, we demonstrate that multiple EV surface proteins can be profiled simultaneously with high sensitivity and specificity. This approach was also successfully applied to similar protocol using cell and serum samples. We further described the development of a micro-size exclusion chromatography method, where we were able to detect EV surface proteins with as little as 10 μL of human serum when combined with immuno-PCR. Overall, these results show that the immuno-PCR method results in rapid detection of multiple EV markers from small sample volumes in a single tube.
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通讯作者：
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