title: Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics.
abstract: 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.
authors.affiliation: Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL, 60208, USA.
authors.forename: Yunqiu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL, 60208, USA.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: McClure
authors.affiliation: Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL, 60208, USA. n-kelleher@northwestern.edu.
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D039903:Actinobacteria / Q000737:chemistry / Q000201:enzymology* / Q000254:growth & development; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010453:Peptide Synthases / Q000032:analysis*; D048630:Polyketide Synthases / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D048630:Polyketide Synthases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: Liquid chromatography; Mass spectrometry; Natural product; Nonribosomal peptide; Polyketide; Proteomics
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