Mycobacterium tuberculosis under starvation

ABSTRACT: Mycobacterium tuberculosis was grown in Middlebrook 7H9 medium supplemented with 0.4% glycerol, 0.085% NaCL, 0.5% BSA and 0.05% Tyloxapol in roller bottle culture (2 rpm at 37°C). To induce starvation, exponentially growing bacteria were washed and resuspended in PBS supplemented with 0.025% Tyloxapol. Cultures were harvested at 0h, 12h and 24h after starvation induction. After harvesting and lysis, proteins were extracted, reduced, alkylated, and digested using trypsin. The peptides were analysed in DDA mode on a Thermo LTQ Orbitrap XL. Data analysis: Thermo raw files were converted into mzXML format using ProteoWizard. The acquired MS2 spectra were searched with OMSSA (2.1.9), X!Tandem (CYCLONE, 2010.12.01.1), and MyriMatch (2.1.114) against an Mtb H37Rv protein database (TubercuList v2.3, April 2011) additionally containing reversed sequences of all proteins in the database. Search parameters were as follows: semi-tryptic peptides (proteolytic cleavage after lysine and arginine unless followed by proline) and up to two missed cleavages were allowed, mass tolerance of the precursor ions was set to 20 ppm. Carbamidomethylation at cysteines was set as a fixed modification and oxidation at methionines as a variable modification. The output of the search engine was processed using PeptideProphet and iProphet. Only peptides at a false discovery rate of less than 1% were taken into consideration for further analysis. For MS1 based label-free quantification the openMS v1.8 framework was used as described by (Weisser et al., 2013). Signals were normalised on peptide feature level such that the median signal in each sample is the same. Abundances of the three most intense peptides were averaged to get a protein abundance value. The same peptides were used for protein quantification across all samples and proteins with less than three peptides were included. Associated RNA-seq data have been deposited to EBI ArrayExpress with accession number E-MTAB-1616.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mycobacterium Tuberculosis H37rv

SUBMITTER: Olga T. Schubert  

PROVIDER: PXD000483 | Pride | 2013-11-22

REPOSITORIES: Pride