PRIDE Assigned Tags:Biological Dataset
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The effect of carbon starvation on the Escherichia coli SLE1 proteome.
The project aims at elucidating the effects of carbon starvation on the qualitative and quantitative composition of the Escherichia coli HT115-derived SLE1 strain proteome as determined by the combination of label-free and metabolic labeling-based proteomics.
Sample Processing Protocol
We used HT115-derived Escherichia coli strain SLE1 auxotrophic for arginine and lysine. The cells were grown in M9 minimal medium (5.8 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl2, 1 mM MgSO4, 0.2% glucose, 0.01% thiamine), supplemented with 0.3 mM of either 12C6-lysine/12C614N4-arginine ('light') or 13C6-lysine/13C615N4-arginine (‘heavy’) amino acids, at 37°C and 150 rpm. Carbon starvation was achieved by incubation of the cells in the medium devoid of amino acids and glucose for 48 hrs. The cells were collected by centrifugation, washed once with cold PBS and lysed in 1X LDS loading buffer (Novex). After estimation of protein concentration, equal quantities of protein, typically ≤400 μg, were processed in accordance with FASP protocol. Briefly, a necessary volume of a reduced protein sample, up to 30 μL, was mixed with 200 µL of 8 M urea in 0.1 M Tris-HCl pH 8.5 and loaded on 10 kDa cut-off spin filters (Millipore). Cysteines were alkylated by 50 mM iodoacetamide in the urea solution for 20 min in the dark. Protein digestion was performed by 15 ng/μL trypsin in 50 mM ammonium bicarbonate for 18 hrs at 37°C. Eluted peptides were desalted on Vivapure C18 micro spin columns (Sartorius Stedim Biotech), desiccated in SpeedVac and dissolved in 10 µL of LC buffer A (0.1% formic acid in water). LC/MS analysis was performed on EASY-nLC 1000 (Thermo Scientific) paired with Q Exactive quadrupole-orbitrap hybrid mass spectrometer (Thermo Scientific). The peptide mixture was separated on EASY-Spray 15 cm × 75 µm 3 µm 100Å C18 PepMap® reverse-phase column (Thermo Scientific) using 150 min three-step water-acetonitrile gradient (0-120 min, 5 → 35% LC buffer B (0.1% formic acid in acetonitrile); 120-140 min, 35 → 50%; 140-145 min, 50 → 90%; hold for 5 min) at 300 nL/min flow rate. The intensities of precursor ions were gauged in positive mode at scan range 400-2,000 m/z, resolution 70,000, automatic gain control (AGC) target 1E6, maximum injection time 100 ms, followed by forwarding 10 most intense ions of a spectrum for MS2 fragmentation and measurement at resolution 17,500, AGC target 5E4, maximum injection time 100 ms, isolation window 2 m/z with 30 sec dynamic exclusion.
Data Processing Protocol
Raw mass spectrometric data were analyzed by Proteome Discoverer v.184.108.40.2068. MS2 spectra were searched against the Escherichia coli Swiss-Prot database using Mascot engine set for 10 ppm precursor mass and 0.02 Da fragment mass tolerances with 2 allowed missed cleavage sites. For labeled samples, the amino acid modifications were as follows: 13C6-lysine (+6.020129 Da) and 13C615N4-arginine (+10.008269 Da) SILAC labels, methionine oxidation (+15.994915 Da) as dynamic, cysteine carbamidomethylation (+57.021464 Da) as static. For unlabeled samples: methionine oxidation and asparigine/glutamine deamidation (+0.984016 Da) as dynamic, cysteine carbamidomethylation as static. False discovery rate (FDR) was calculated using Percolator with 0.01 strict and 0.05 relaxed target cut-off values.
No PubMed ID is available for this publication since this journal is not indexed in PubMed. However, a DOI is provided: 10.7287/PEERJ.PREPRINTS.1572V1
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