Department of Biological Sciences, Columbia University, New York, NY 10027;
Department of Systems Biology, Columbia University, New York, NY 10032.
Proc Natl Acad Sci U S A. 2020 Oct 27;117(43):26710-26718. doi: 10.1073/pnas.2002650117. Epub 2020 Oct 9.
Large-scale proteomic methods are essential for the functional characterization of proteins in their native cellular context. However, proteomics has lagged far behind genomic approaches in scalability, standardization, and cost. Here, we introduce in vivo mRNA display, a technology that converts a variety of proteomics applications into a DNA sequencing problem. In vivo-expressed proteins are coupled with their encoding messenger RNAs (mRNAs) via a high-affinity stem-loop RNA binding domain interaction, enabling high-throughput identification of proteins with high sensitivity and specificity by next generation DNA sequencing. We have generated a high-coverage in vivo mRNA display library of the proteome and demonstrated its potential for characterizing subcellular localization and interactions of proteins expressed in their native cellular context. In vivo mRNA display libraries promise to circumvent the limitations of mass spectrometry-based proteomics and leverage the exponentially improving cost and throughput of DNA sequencing to systematically characterize native functional proteomes.
大规模的蛋白质组学方法对于在天然细胞环境下对蛋白质的功能进行特征描述是必不可少的。然而，蛋白质组学在可扩展性、标准化和成本方面远远落后于基因组学方法。在这里，我们介绍了体内 mRNA 展示技术，它将各种蛋白质组学应用转化为 DNA 测序问题。通过高亲和力的茎环 RNA 结合域相互作用，将体内表达的蛋白质与其编码的信使 RNA（mRNA）连接起来，通过下一代 DNA 测序以高灵敏度和特异性高通量鉴定蛋白质。我们已经生成了一个高覆盖率的体内 mRNA 展示文库，展示了其在描述天然细胞环境中表达的蛋白质的亚细胞定位和相互作用方面的潜力。体内 mRNA 展示文库有望克服基于质谱的蛋白质组学的局限性，并利用 DNA 测序成本和通量呈指数级提高的优势，系统地描述天然功能蛋白质组。
