LC-MS metabolite identification in extracts from different zones of 79NG-agar culture of Streptomyces coelicolor A3(2) ∆scbA-atrAOE strain upon GBL addition

The experimental project at aim of this dataset was to identify, via LC-MS, compounds related to actinorhodin biosynthesis pathway from different zones of 79NG-agar culture of Streptomyces coelicolor A3(2) ∆scbA-atrAOE mutant strain. The zones differed by γ-butyrolactone (GBL) presence as follows: samples A — control zone (cut out after 6h of growth, before GBLs addition) samples B — zone of GBLs diffusion samples C — outer zone (no GBLs diffusion after addition) Methodology was as follows: Metabolite extraciton was performed via ethyl acetate from solid agar plates. After centrifugation the soluble fraction was evaporated in a rotary evaporator. Samples were reconstituted in 50:50ACN:H2O+0.1%FA at a ratio of 0.5g of agar mass to 1mL of solution. Prior to analysis each sample was dilluted 5x with a 0.1%FA solution to a final concentration of 10%ACN.LC-MS was performed on an M-Class Acquity UPLC connected to a Synapt XS HDMS. Mobile phase A consisted of H2O+0.1% FA, while mobile phase B of ACN+0.1% FA. 3µL of each sample were injected and a 15min 5–85%B linear gradient was applied on a C18 BEH 1mm x 100mm analytical column for sample separation at 50µL/min. Data were collected in ESI+ using DDA mode, with MS and MS/MS scan rate of 0.1s, both in the 100–1700 m/z range. The top 5 precursors from each MS scan were selected for MS/MS with 1 scan allowed per transition and a dynamic exclusion window of 24s. Collision energy ramp determined specifically for each m/z (start LM, HM: 15–55V; end LM, HM: 20–65V) was applied on the trap cell. To minimize background related MS/MS, a precursor exclusion list was generated from precursor peaks present in blank runs performed prior to proper analysis and applied (95 precursors, exclusion criteria: ±20 ppm and ±12s RT). Source conditions were fine-tuned. A Leucine-Enkephalin solution was acquired in-parallel as lockmass, and correction was applied in-acquisition. Three independent biological replicates were analyzed (n=3), 9 samples in total.For qualitative analysis raw files were processed in PLGS v3.0.3 (Waters). Obtained spectra were exported as .mgf files. Queries were limited to top 20 ions using MSConvert (Chambers, 2012), and then matched against GNPS libraries (Wang, et al., 2016). The library search parameters were as follows; Precursor Ion Mass Tolerance: 0.01Da, Fragment Ion Mass Tolerance: 0.01Da, Score Threshold: 0.5, Min. Matched Peaks: 4. The 17Da window option filtering was enabled, while the 50Da window filtering option was disabled. Analogs were not considered. The Top Hits list was analyzed for compounds related to actinorhodin biosynthesis (5 out of 100 hits). For actinorhodin biosynthesis compounds not present in GNPS libraries, we manually investigated the .mgf files to compare with MS/MS spectra available in literature (Marshall & Carlson, 2023).For relative quantitative analysis, an MS1 precursor transition list was generated based on the qualitative workflow results and imported into Skyline (Adams, et al., 2020). The MS1 level was filtered to .mzML format from each of the raw files using MSConvert. In Skyline, XIC traces were extracted from the .mzML files using a 0.01 m/z window around 0.5min of the expected RT (the actual widest RT apex window was 2.97s) according to the imported transition list. Peaks were then integrated and their areas were TIC normalized across samples.Adams, K., B, P., Bose, N., Dubois, L., St John-Williams, L., Perrott, K., . . . Thompson, J. (2020). Skyline for Small Molecules: A Unifying Software Package for Quantitative Metabolomics. J Proteome Res., 19(4), 1447-1458. doi:10.1021/acs.jproteome.9b00640Chambers, M. M. (2012). A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotechnol 30, 918–920.Marshall, A. P., & Carlson, E. E. (2023). Metabolomics Reveals a “Trimeric” γ-Actinorhodin from Streptomyces coelicolor M145. ChemBioChem, 24(7), e202200757. doi:doi.org/10.1002/cbic.202200757Wang, M., Carver, J. J., Phelan, V. V., Sanchez, L. M., Garg, N., Peng, Y., . . . al., e. (2016). Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nature Biotechnology, 34(8), 828.