Targeted proteomics assays are applied to specific hypotheses via selected-reaction monitoring (SRM) mass spectrometry methods that accurately quantify a select group of proteins. This approach has been applied to determine pathway bottlenecks, compare enzyme homologs for a specific enzymatic step, and to track dynamic regulation of protein levels engineered microbes. For instance, application of targeted proteomics methods helped identify protein-associated bottlenecks in the mevalonate pathway, resulting in over 300% improvement in the final product levels. This technique is also used to quantify protein levels for tool genetic development studies such as engineered promoter and ribosome-binding site (RBS) variants. More recently, targeted proteomic methods have been optimized for greater throughput (over 250 samples/day) as well as used to characterize and quantify stable post-translational modifications for engineered microbes. High-throughput targeted proteomic methods for engineered microbes utilize laboratory automation to reduce sample preparation variability and leverage tight integration with the ABF ICE sequence/strain repository platform and Experiment Data Depot (EDD). The targeted proteomic capability supports the ABF through rapid testing of engineered microbes to reduce the number of DBTL cycles required to successfully produce target molecules. Targeted proteomic assays are limited to quantify fewer than 75 proteins in high-throughput applications, but can accurately quantify hundreds of proteins for lower throughput experiments. The method is also requires that unique peptides for a specific protein can be targeted. Each protein and peptide has different limits of detection, so sensitivity ranges will vary depending on the experiment. The targeted proteomics methods are available to ABF researchers as well as ABF CRADA projects.
References and Additional Information:
Dahl, Robert H., et al. “Engineering dynamic pathway regulation using stress-response promoters.” Nature biotechnology 31.11 (2013): 1039.
George, Kevin W., et al. “Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli.” Metabolic engineering (2018).
Brunk, Elizabeth, et al. “Characterizing strain variation in engineered E. coli using a multi-omics-based workflow.” Cell systems 2.5 (2016): 335-346.
Lawrence Berkeley National Laboratory
Pacific Northwest National Laboratory