Computational protocol: Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

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Protocol publication

[…] The proteomic sample processing and data analysis in Additional file : Table S8 has been reported previously at ORNL [, ]. We here described the method for GLBRC proteomic data shown in Additional file : Table S9. Ten milliliters of Z. mobilis cells was collected at different growth conditions by centrifugation at 15,000g for 3 min, and quickly frozen in ethanol/dry ice bath and stored at − 80 °C until use. Cells were lysed by suspension in 6 M guanidine hydrochloride (GnHCl), followed by addition of MeOH to 90%. Samples were centrifuged at 15,000g for 5 min. Supernatant was discarded and pellets were allowed to dry for ~ 5 min. Pellets were resuspended in 200 µL 8 M urea, 100 mM Tris (pH 8.0), 10 mM TCEP, and 40 mM chloroacetamide, then diluted to 2 M urea in 50 mM Tris (pH 8). Trypsin was added at an estimated 50:1 (protein to enzyme) ratio, and samples were incubated overnight at ambient temperature. Each sample was desalted over a PS-DVB solid phase extraction cartridge and dried down. Peptide mass was assayed with the peptide colorimetric assay.For each analysis, 2 µg of peptides was loaded onto a 75 µm i.d. 30-cm-long capillary with an imbedded electrospray emitter and packed with 1.7 µm C18 BEH stationary phase. The mobile phases used were A: 0.2% formic acid and B: 0.2% formic acid in 70% acetonitrile. Peptides were eluted with in increasing gradient of acetonitrile from 0 to 53% B over 75 min followed by a 5 min 100% B wash and a 10-min equilibration in 0% B []. Eluting peptides were analyzed with an Orbitrap Fusion Lumos. Survey scans were performed at R = 60,000 with wide isolation analysis of 300–1350 mz. Data-dependent top speed (2 s) MS/MS sampling of peptide precursors was enabled with dynamic exclusion set to 45 s on precursors with charge states 2–6. MS/MS sampling was performed with 1.6 Da quadrupole isolation, fragmentation by HCD with NCE of 30, analysis in the Orbitrap at R = 15,000 with max inject time of 22 ms, and AGC target set to 2 × 105.Raw files were analyzed using MaxQuant Spectra were searched using the Andromeda search engine against a target decoy database generated in house. As described previously [, ], label-free quantitation and match between runs were toggled on and the number of peptides measurements for each protein was set to 1. Default parameters were used for all other analysis parameters. Peptides were grouped into subsumable protein groups and filtered to 1% FDR, based on target decoy approach. […]

Pipeline specifications

Software tools MaxQuant, Andromeda
Application MS-based untargeted proteomics
Organisms Zymomonas mobilis
Chemicals Nucleotides, Oxygen, Xylose