Fig. 4
Shotgun Proteomics Identifies Enriched Proteins in cydC-D86G Cells Upon [EMIM]OAc Treatment. a Strains harboring the limonene production plasmid (JBEI-6409) and a plasmid-borne copy of cydC-D86G [pTE42/Ptrc-cydD-cydC-D86G] were prepared for whole genome proteomic analysis (“Methods”). Samples were harvested immediately before induction with 1 µM IPTG and 48 h after induction. Proteins at least 2 × enriched were categorized into functional categories (COG, Cluster of Orthologous Groups) (“Methods”). White columns; distribution of COG terms from the E. coli genome [29]. Gray columns; subset of recovered proteins from shotgun proteomics binned into COGs and plotted as the percent of all proteins recovered. Blue striped bars; COG distribution from DH1 treated with 100 mM [EMIM]OAc plotted as the percent of enriched proteins. Black bars; COG distribution from DH1 cydC-D86G treated with 100 mM [EMIM]OAc plotted as the percent of enriched proteins. b Candidate effector proteins enriched at least 2× were validated with quantitative peptide-area proteomics. The same experimental regimen and cells used in a were used to confirm protein abundance. The fold enrichment (log2) of unique peptides are plotted against their normalized significance (P) values. COG descriptions. [C], Energy production and conversion. [E], Amino Acid metabolism and transport. [G], Carbohydrate metabolism and transport. [H], Coenzyme metabolism. [I], Lipid metabolism. [J], Translation. [K], Transcription. [M], Cell wall/membrane/envelope biogenesis. [O], Post-translational modification, protein turnover, chaperone function. [P]. Inorganic ion transport and metabolism. [S], Function Unknown. [T], Signal Transduction. [U], Intracellular Trafficking and Secretion