Reich Lothar Luther, Dutta Sanjib, Keating Amy E
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Rm 68-622A, Cambridge, MA, 02139, USA.
Methods Mol Biol. 2016;1414:233-47. doi: 10.1007/978-1-4939-3569-7_14.
Library methods are widely used to study protein-protein interactions, and high-throughput screening or selection followed by sequencing can identify a large number of peptide ligands for a protein target. In this chapter, we describe a procedure called "SORTCERY" that can rank the affinities of library members for a target with high accuracy. SORTCERY follows a three-step protocol. First, fluorescence-activated cell sorting (FACS) is used to sort a library of yeast-displayed peptide ligands according to their affinities for a target. Second, all sorted pools are deep sequenced. Third, the resulting data are analyzed to create a ranking. We demonstrate an application of SORTCERY to the problem of ranking peptide ligands for the anti-apoptotic regulator Bcl-xL.
文库方法被广泛用于研究蛋白质-蛋白质相互作用，高通量筛选或选择后测序能够鉴定出针对蛋白质靶标的大量肽配体。在本章中，我们描述了一种称为“SORTCERY”的程序，它可以高精度地对文库成员与靶标的亲和力进行排序。SORTCERY遵循一个三步方案。首先，利用荧光激活细胞分选（FACS）根据酵母展示肽配体与靶标的亲和力对其文库进行分选。其次，对所有分选的文库池进行深度测序。第三，分析所得数据以生成一个排序。我们展示了SORTCERY在对抗凋亡调节因子Bcl-xL的肽配体进行排序问题上的应用。
