Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, Georgia, USA.
Center for Biotechnology and Genomic Medicine, Medical College of Georgia, Augusta University, Augusta, Georgia, USA.
J Extracell Vesicles. 2022 Jun;11(6):e12239. doi: 10.1002/jev2.12239.
The extracellular vesicle exosome mediates intercellular communication by transporting macromolecules such as proteins and ribonucleic acids (RNAs). Determining cargo contents with high accuracy will help decipher the biological processes that exosomes mediate in various contexts. Existing methods for probing exosome cargo molecules rely on a prior exosome isolation procedure. Here we report an in situ labelling approach for exosome cargo identification, which bypasses the exosome isolation steps. In this methodology, a variant of the engineered ascorbate peroxidase APEX, fused to an exosome cargo protein such as CD63, is expressed specifically in exosome-generating vesicles in live cells or in secreted exosomes in the conditioned medium, to induce biotinylation of the proteins in the vicinity of the APEX variant for a short period of time. Mass spectrometry analysis of the proteins biotinylated by this approach in exosomes secreted by kidney proximal tubule-derived cells reveals that oxidative stress can cause ribosomal proteins to accumulate in an exosome subpopulation that contains the CD63-fused APEX variant.
细胞外囊泡（exosome）通过转运蛋白质和核糖核酸（RNAs）等大分子来介导细胞间通讯。准确确定货物内容将有助于破译 exosome 在各种情况下介导的生物学过程。现有的探测 exosome 货物分子的方法依赖于 exosome 分离程序。在这里，我们报告了一种用于 exosome 货物鉴定的原位标记方法，该方法绕过了 exosome 分离步骤。在这种方法中，一种工程化的抗坏血酸过氧化物酶 APEX 的变体与 exosome 货物蛋白（如 CD63）融合，在活细胞中产生 exosome 的囊泡或在条件培养基中分泌的 exosome 中特异性表达，以在短时间内诱导 APEX 变体附近蛋白质的生物素化。用这种方法在肾近端小管衍生细胞分泌的 exosome 中生物素化的蛋白质的质谱分析表明，氧化应激可导致核糖体蛋白在含有融合 CD63 的 APEX 变体的 exosome 亚群中积累。
