| Metadata |
| datasetIdentifier | PASS00655 |
| datasetType | SWATH |
| submitter | Olga Schubert <olga.schubert@gmail.com> |
| submitter_organization | ETH Zurich |
| lab_head_full_name | Ruedi Aebersold |
| lab_head_email | aebersold@imsb.biol.ethz.ch |
| lab_head_organization | ETH Zurich |
| lab_head_country | Switzerland |
| datasetTag | Mtb_ALF-SWATH |
| datasetTitle | Absolute proteome composition and dynamics during dormancy and resuscitation of Mycobacterium tuberculosis by SWATH MS |
| publicReleaseDate | 2015-05-05 00:00:00 |
| finalizedDate | 2016-08-11 00:15:27 |
| summary | Life science research faces an increasing demand for absolute quantification of biomolecules to determine the molecular composition of a cell and to support mathematical modelling of biochemical processes. Here, we implemented a universally applicable, label-free strategy to estimate absolute cellular protein concentrations on a proteome-wide scale based on SWATH mass spectrometry. We applied this strategy to study proteomic reorganisation in the human pathogen Mycobacterium tuberculosis during exponential growth, hypoxia-induced dormancy and resuscitation. The resulting data set covering >2000 proteins reveals how protein biomass is distributed among cellular functions and dynamically remodelled in response to hypoxic stress. We found that the DosR regulon contributes 20% to the entire cellular protein content during dormancy, whereas the fraction of ribosomal proteins remains largely unchanged at 5-7%. Knowledge of protein copies/cell furthermore allowed us to translate effects of protein regulation into changes in maximal enzymatic reaction velocities, enhancing our understanding of metabolic adaptations. |
| contributors | Schubert, OT, Ludwig, C, Gengenbacher, M, Kaufmann, SHE, and Aebersold, R |
| publication | Schubert, OT, Ludwig, C, Kogadeeva M, Zimmermann, M, Rosenberger, G, Gengenbacher, M, Gillet, LC, Collins, BC, Roest, HL, Kaufmann, SHE, Sauer, U, and Aebersold, R, Absolute proteome composition and dynamics during dormancy and resuscitation of Mycobacterium tuberculosis by SWATH MS, Cell Host & Microbe, Jul 8;18(1):96-108 (PMID: 26094805) |
| growth | Mtb H37Rv (ATCC #27294) and M. bovis BCG was grown in Dubos broth enriched with Dubos Medium Albumin (Becton Dickinson) at 37°C. |
| treatment | Hypoxia experiments were started by diluting exponentially growing bacteria (OD600=0.4, “day 0”) to an OD600 of 0.005 as described earlier (Lim et al., 1999; Wayne and Hayes, 1996). The tightly sealed glass vials with a fixed headspace-culture ratio of 1:2 were kept closed until harvesting to prevent exposure to external oxygen. Samples were taken at 5, 10 and 20 days of the hypoxic time course (“day 5”, “day 10” and “day 20”). After sampling at day 20, remaining glass vials were opened and cultures were transferred into new vessels for aerated incubation at 80 rpm and 37°C. Further time points were sampled from the re-aerated cultures after 6 and 48 hours (“day 20+6h” and “day 20+48h”). |
| extraction | Bacteria from 5 Wayne flasks (87.5 ml) were harvested by centrifugation at 3000 g for 10 min and washed with ice-cold phosphate-buffered saline. Pellets were then resuspended in lysis buffer containing 8 M urea and 0.1% RapiGest, (#186001861, Waters) in 0.1 M ammonium bicarbonate buffer. The cell suspension was thoroughly vortexed and incubated at room temperature for 10 min while shaking at 1000 rpm. Subsequently, bacilli were subjected to three 10-min cycles of sonication at 4°C (100% output, 50% intervals, Branson Sonifier 450, Emerson) and additionally disrupted by three 10-min cycles of bead beating at 4°C using glass beads with a diameter of 0.5 mm (SIGMA #G8772). After each cycle, lysates were centrifuged for 10 min at 16,000 g and fresh lysis buffer was added. In total, 250 µl of lysis buffer was added. Protein lysates were sterile filtered twice for decontamination and protein concentration was determined using a BCA assay according to the manufacturer’s protocol (#23227, Thermo Fisher Scientific). From each sample only 50 µg of protein was used for subsequent steps. |
| separation | |
| digestion | Protein disulfide bonds were reduced by adding 5 mM tris(2-carboxyethyl)phosphine (TCEP) and incubating for 30 min at 30°C. Next, the free cysteine residues were alkylated by adding 40 mM iodoacetamide and incubating for 60 min in the dark at room temperature. Subsequently, the samples were diluted with 0.05 M ammonium bicarbonate buffer to reach a urea concentration of 6.7 M. 0.5 µg LysC (#125-05061, Wako) was added to each sample (w/w 1:100) and incubated at 30°C for 6 hours. Then the samples were further diluted to a urea concentration <2 M and 1 µg of sequencing-grade modified trypsin (#608-274-4330, Promega) was added (w/w 1:50). The samples were incubated over night at 30°C with gentle shaking at 300 rpm. At this step isotopically labelled synthetic reference peptides (AQUA QuantPro, Thermo Fisher Scientific) for absolute quantification of the 30 anchor proteins were added to the samples in concentrations similar to the endogenous peptide. To stop the tryptic digest and to precipitate RapiGest the pH was lowered to 2 using trifluoro acetic acid (final concentration of ~1%) followed by an incubation for 30 min at 37°C with shaking at 500 rpm. The water-immiscible degradation product of RapiGest was pelleted by centrifugation at 16,000 g for 10 min. The cleared peptide solution was desalted with C18 MicroSpin columns (The Nest Group, 5-60 µg loading capacity). Prior to use, the C18 columns were activated with 100% methanol, followed by 80% acetonitrile (ACN)/0.1% TFA, followed by equilibration with 2% ACN/0.1% TFA. After loading the sample, the columns were washed with 2% ACN/0.1% TFA. Finally, peptides were eluted with 40% ACN/0.1% TFA, dried under vacuum, and resolubilised in 46 µl 2% ACN/0.1% FA and 4 µl iRT peptide mix (RT-kit WR, Biognosys) to a final concentration of 1 mg/ml. The iRT peptides are important to allow determination of system-independent retention times (iRT) for each peptide relative to these calibration peptides as recently described by Escher and colleagues (Escher et al., 2012). |
| acquisition | The TripleTOF 5600 mass spectrometer (AB Sciex) was coupled to a nanoLC 1Dplus system (Eksigent) and the chromatographic separation of the peptides was performed on a 20-cm emitter (75 μm inner diameter, #PF360-75-10-N-5, New Objective) packed in-house with C18 resin (Magic C18 AQ 3 μm diameter, 200 Å pore size, Michrom BioResources). A linear gradient from 2-35% solvent B (98% ACN/0.1% FA) was run over 120 min at a flow rate of 300 nl/min. The mass spectrometer was operated in SWATH mode as described previously (Gillet et al., 2012). Briefly, acquisition of a 100-ms survey scan was followed by acquisition of 32 fragment ion spectra from 32 precursor isolation windows (swaths) of 26 m/z each. The swaths were overlapping by 1 m/z and thus cover a range of 400-1200 m/z. The SWATH MS2 spectra were recorded with an accumulation time of 100 ms and cover 100-2000 m/z. The collision energy for each window was determined according to the calculation for a charge 2+ ion centred upon the window with a spread of 15V. |
| informatics | The SWATH data was analysed using OpenSWATH (Röst et al., 2014) with the following parameters: Chromatograms were extracted with 50 ppm around the expected mass of the fragment ions and with an extraction window of +/-5 min around the expected retention time. The best models to separate true from false positives (per run) were determined by pyprophet with 10 cross-validation runs (Teleman et al., 2014). The runs were subsequently aligned with a target FDR of 0.01 and a maximal FDR of 0.05 for aligned features (Röst et al., unpublished). In the absence of a confidently identified feature, the peptide and protein intensities were obtained by integration of the respective background signal at the expected peptide retention time (Röst et al., unpublished). To reduce the size of the output data and remove low-quality features, a filtering step was introduced to only keep the 10 most intense peptides per protein and only features that were identified with an FDR of <0.01 in all replicates of at least one condition. |
| instruments | AB Sciex TripleTOF 5600+ |
| species | Mycobacterium tuberculosis, Mycobacterium bovis BCG |
| massModifications | C+57.021464 |
