Biomolecular Measurement Division, National Institute of Standards and Technology , Gaithersburg, Maryland 20899, United States.
Institute for Bioscience and Biotechnology Research , Rockville, Maryland 20850, United States.
Anal Chem. 2017 Oct 17;89(20):11070-11075. doi: 10.1021/acs.analchem.7b03119. Epub 2017 Oct 5.
The increasing interest in extracellular vesicles (EVs) research is fueled by reports indicating their unique role in intercellular communication and potential connection to the development of common human diseases. The unique role assumes unique protein and nucleic acid cargo. Unfortunately, accurate analysis of EVs cargo faces a challenge of EVs isolation. Generally used isolation techniques do not separate different subtypes of EVs and even more, poorly separate EVs from non-EVs contaminants. Further development of EVs isolation protocols urgently needs a quantitative method of EVs purity assessment. We report here that multiple reaction monitoring assay using internal standards carrying peptides for quantification of EVs and non-EVs proteins is a suitable approach to assess purity of EVs preparations. As a first step in potential standardization of EVs isolation, we have evaluated polymer-based precipitation techniques and compared them to traditional ultracentrifugation protocol.
细胞外囊泡 (EVs) 的研究日益受到关注，这是因为有报道称 EVs 在细胞间通讯中具有独特的作用，并可能与常见人类疾病的发展有关。这种独特的作用假定了独特的蛋白质和核酸货物。不幸的是，EVs 货物的准确分析面临着 EVs 分离的挑战。通常使用的分离技术不能分离不同亚型的 EVs，甚至更不能将 EVs 与非 EVs 污染物有效分离。EVs 分离方案的进一步发展迫切需要一种 EVs 纯度评估的定量方法。我们在这里报告，使用带有肽的内标进行多重反应监测分析，是定量检测 EVs 和非 EVs 蛋白质的一种合适方法，可用于评估 EVs 制剂的纯度。作为 EVs 分离潜在标准化的第一步，我们已经评估了基于聚合物的沉淀技术，并将其与传统的超速离心法进行了比较。
