Quantitative Proteomics of Recombinant Streptomyces

Quantitative proteomics was used to analyse the expression of catechol dioxygenases and the expression profile of BGC4 in recombinant strains of Streptomyces sp. MBT84 as described previously^{45 (link)}. Briefly, the desalted peptide solution was separated on an UltiMate 3000 RSLCnano system (Thermo Scientific) set in a trap-elute configuration, coupled to QExactive HF (Thermo Scientific) mass spectrometer. The LC system used a Waters nanoEase M/Z Symmetry C₁₈ trap column (5 µm, 100 Å, 180 µm × 20 mm) for peptide loading/retention, and Waters nanoEase M/Z HSS T3 C₁₈ analytical column (1.8 µm, 100 Å, 75 µm × 250 mm) for peptide separation. The MS was operated in positive mode with data-dependent acquisition and default charge of 2. Raw LC-MS/MS files were analysed using MaxQuant software (v1.6.17.0)^{57 (link)} with label-free quantification (LFQ) method applied. Proteins were considered significantly altered in expression when FDR-adjusted p < 0.1 were obtained.