1] Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts, USA. [2] Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] Department of Genetics, Harvard University Medical School, Boston, Massachusetts, USA. [4] Department of Medicine, Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, USA. [5] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. [6].
1] Department of Medicine, Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, USA. [2] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
Nat Protoc. 2014 Jan;9(1):90-103. doi: 10.1038/nprot.2013.167. Epub 2013 Dec 12.
Parallel analysis of translated open reading frames (ORFs) (PLATO) can be used for the unbiased discovery of interactions between full-length proteins encoded by a library of 'prey' ORFs and surface-immobilized 'bait' antibodies, polypeptides or small-molecular-weight compounds. PLATO uses ribosome display (RD) to link ORF-derived mRNA molecules to the proteins they encode, and recovered mRNA from affinity enrichment is subjected to analysis using massively parallel DNA sequencing. Compared with alternative in vitro methods, PLATO provides several advantages including library size and cost. A unique advantage of PLATO is that an alternative reverse transcription-quantitative PCR (RT-qPCR) protocol can be used to test binding of specific, individual proteins. To illustrate a typical experimental workflow, we demonstrate PLATO for the identification of the immune target of serum antibodies from patients with inclusion body myositis (IBM). Beginning with an ORFeome library in an RD vector, the protocol can produce samples for deep sequencing or RT-qPCR within 4 d.
可将翻译后的开放阅读框（ORF）（PLATO）进行平行分析，用于无偏发现文库中全长“诱饵”ORF 编码的蛋白质与表面固定的“诱饵”抗体、多肽或小分子化合物之间的相互作用。PLATO 使用核糖体展示（RD）将 ORF 衍生的 mRNA 分子与它们编码的蛋白质连接起来，并从亲和富集中回收的 mRNA 进行大规模平行 DNA 测序分析。与替代的体外方法相比，PLATO 具有多个优势，包括文库的大小和成本。PLATO 的一个独特优势是可以使用替代的反转录定量聚合酶链反应（RT-qPCR）方案来测试特定单个蛋白质的结合。为了说明典型的实验工作流程，我们展示了 PLATO 用于鉴定包涵体肌炎（IBM）患者血清抗体的免疫靶标。从 RD 载体中的 ORFeome 文库开始，该方案可以在 4 天内产生用于深度测序或 RT-qPCR 的样本。
