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.
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Acknowledgement
This work was supported by National Institute of General Medical Sciences of the National Institutes of Health under award number R01 GM067725. We thank the Agricultural Research Service, US Department of Agriculture for providing the bacterial strains.
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Chen, Y., McClure, R.A., Kelleher, N.L. (2016). Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics. In: Evans, B. (eds) Nonribosomal Peptide and Polyketide Biosynthesis. Methods in Molecular Biology, vol 1401. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3375-4_9
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DOI: https://doi.org/10.1007/978-1-4939-3375-4_9
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