Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA.
Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA; Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA.
J Proteomics. 2021 Oct 30;249:104359. doi: 10.1016/j.jprot.2021.104359. Epub 2021 Aug 25.
Absolute quantification with mass spectrometry and isotope labeled internal standards has found broad applications in biomedical research. In the present research, it was used for developing and evaluating a new affinity-based approach to isolate extracellular vesicles (EVs) from human plasma. First, a phage display peptide library was screened against EVs as a bait and absolute quantification of multiple proteins helped to select the best bait available. Then, absolute quantification was used to evaluate the efficiency of affinity chromatography on peptide-Sepharose. In summary, we have demonstrated that peptides with affinity to EVs selected from phage library screening can be valuable ligands for EVs isolation. SIGNIFICANCE: Extracellular vesicles (EVs) have an important role in intercellular communication for all cell types. This makes EVs a promising new type of therapeutics capable to deliver drugs to specific sites with no off-target side effects. However, their isolation, and correct assignment of their biological function and properties remains an obscure field of research. In this study, we proposed to use MRM quantitation of a pattern of EVs and non-EVs proteins to develop a purification protocol based on affinity peptides selected from phage library screening. MRM quantification of EVs proteins can also help in identifying those that are subpopulation specific markers for further target-specific isolation.
绝对定量与质谱和同位素标记内标已在生物医学研究中得到广泛应用。在本研究中，我们使用它来开发和评估一种新的基于亲和的方法，从人血浆中分离细胞外囊泡 (EVs)。首先，我们用噬菌体展示肽文库筛选 EVs 作为诱饵，并用绝对定量的多种蛋白质来选择最佳的可用诱饵。然后，我们用绝对定量来评估肽-琼脂糖亲和层析的效率。总之，我们已经证明，从噬菌体文库筛选中选择的对 EVs 具有亲和力的肽可以作为 EVs 分离的有用配体。意义：细胞外囊泡 (EVs) 在所有细胞类型的细胞间通讯中起着重要作用。这使得 EVs 成为一种很有前途的新型治疗药物，能够将药物输送到特定的靶点，而没有脱靶的副作用。然而，它们的分离以及对其生物学功能和特性的正确分配仍然是一个模糊的研究领域。在这项研究中，我们提出使用 EVs 和非-EVs 蛋白的 MRM 定量来开发一种基于从噬菌体文库筛选中选择的亲和肽的纯化方案。EVs 蛋白的 MRM 定量也有助于识别那些是特定亚群的标记物，以便进一步进行靶向特异性分离。
