Chen Yunqiu, McClure Ryan A, Kelleher Neil L
Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL, 60208, USA.
Methods Mol Biol. 2016;1401:135-47. doi: 10.1007/978-1-4939-3375-4_9.
Liquid chromatography-mass spectrometry (LC-MS)-based proteomics is a powerful technique for the profiling of protein expression in cells in a high-throughput fashion. Herein we report a protocol using LC-MS/MS-based proteomics for the screening of enzymes involved in natural product biosynthesis, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) from bacterial strains. Taking advantage of the large size of modular NRPSs and PKSs (often >200 kDa), size-based separation (SDS-PAGE) is employed prior to LC-MS/MS analysis. Based upon the protein identifications obtained through software search, we can accurately pinpoint the expressed NRPS and/or PKS gene clusters from a given strain and growth condition. The proteomics screening result can be used to guide the discovery of potentially new nonribosomal peptide and polyketide natural products.
基于液相色谱-质谱联用（LC-MS）的蛋白质组学是一种强大的技术，可用于高通量分析细胞中的蛋白质表达情况。在此，我们报告一种基于LC-MS/MS蛋白质组学的方案，用于筛选参与天然产物生物合成的酶，例如来自细菌菌株的非核糖体肽合成酶（NRPS）和聚酮合酶（PKS）。利用模块化NRPS和PKS的大尺寸（通常>200 kDa），在进行LC-MS/MS分析之前采用基于大小的分离方法（SDS-PAGE）。基于通过软件搜索获得的蛋白质鉴定结果，我们可以准确地从给定菌株和生长条件中找出表达的NRPS和/或PKS基因簇。蛋白质组学筛选结果可用于指导发现潜在的新型非核糖体肽和聚酮天然产物。
