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.