Phosphoproteomics COMMSBIO-18-0285B

For Phosphoproteomics, Mtb strains (Mtb H37Rv Wild type and mutants MtbΔdosR, MtbΔdosS, and MtbΔdosT) were cultured in liquid media with shaking (aerobic, OD595=0.3) or left standing (hypoxic, day 1 to day 30) as described. The cultures were harvested by centrifugation followed by washing in 7H9 media and resuspension in lysis buffer. Bacterial cells were sonicated by bead beating, and after filtration through 0.22 ìm filters; clear lysates were used in downstream procedures. The lysates were reduced with 10 mM dithiothreitol followed by alkylation with 15 mM iodoacetamide and acetone precipitation. The protein pellets were washed in acetone/water (80/20), followed by evaporation of acetone and reconstitution in 200 μL of modified urea lysis buffer (5 M urea, 150 mM NaCl, 50 mM Tris-HCl pH 8.0). Protein concentration was estimated by the Bradford assay (Bio-Rad), and 1 mg of protein was digested with trypsin for 16 h at 37°C followed by enzyme inactivation with 10 μL trifluoroacetic acid (TFA). Next, Phosphopeptide enrichment using TiO2 chromatography was performed. The trypsin-digested peptides were enriched for phosphopeptides using Titansphere TiO2 tips (Thermo scientific USA). Phosphopeptides were serially eluted in 5 % NH4OH in water, 5 % pyrrolidine in acetonitrile, and 60 % acetonitrile in water. The three elutions were pooled together, neutralized with 50 % acetic acid, and dried. Samples were reconstituted in 50 μL 0.03 % TFA. Each enriched sample was desalted using a Stage Tip (ThermoFisher) per the vendor protocol. Peptides were dried and reconstituted in 70 μL of 0.03 % TFA prior to analysis. Next, Mass spectrometry was performed as following. The phosphopeptide-enriched samples were analyzed by mass spectroscopy in collaboration with MS Bioworks. The samples were analyzed by nanoLC-MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive using a 2 h reverse phase gradient. The phosphopeptides were loaded onto a trapping column and eluted over a 75 μm analytical column at 350 nL min-1; both columns were packed with Jupiter Proteome resin (Phenomenex). The mass spectrometer was operated in data-dependent mode, with the Orbitrap operating at 60,000 full width at half maximum (FWHM) and 17,500 FWHM for MS and MS/MS, respectively. The fifteen most abundant ions were selected for MS/MS. The data analyses and phosphoproteins were detected as below. The proteins expressed during onset of hypoxia, i.e., at the early stage of transition during aerobic to hypoxic conditions, were identified by mass spectroscopy. Mascot DAT files were parsed into Scaffold software for validation, filtering and to create a non-redundant list per sample. Data were filtered using at 1 % protein and peptide FDR and requiring at least one unique peptide per protein. Scaffold results were imported into Scaffold PTM in order to assign site localization probabilities using A-score. A minimum localization probability filter of 50 % was applied for the analysis.