For accurate mass measurements

For accurate mass measurements find more the lock mass option was enabled in MS mode and the polydimethylcyclosiloxane (PCM) ions generated in the electrospray process from ambient air (protonated (Si(CH3)2O)6; m/z 445.120025) were used for internal recalibration during the analysis [51]. Target ions already selected for MS/MS were dynamically excluded for 30 seconds. General mass spectrometry conditions were: electrospray voltage, 1.9 kV Ion selection threshold was 500 counts for MS/MS, an activation Q-value of 0.25 and activation time of 30 ms was also applied for MS/MS. The obtained data

was searched against the publicly available Tuberculist database version R10 http://​genolist.​pasteur.​fr/​TubercuList/​ using MASCOT software version 2.1 (Matrix Science, UK). The database was in-house modified to include reversed sequences of the original ORFs in order to determine false-positive thresholds of the Mascot identification engine [52]. Tuberculist was preferred over secondary annotations performed by independent institutes because previous data from our group demonstrated Quisinostat chemical structure that the Tuberculist annotation appear to be more reliable [33]. The criteria for the Mascot search were as follows: Cysteine carbamidomethylation was set as fixed modification,

methionine oxidation and N-acetylation (protein) as variable modifications. Up to 3 missed cleavages were allowed. Peptide

(precursor) ion mass tolerance was 15 ppm, and the fragment ion tolerance was 0.5 Da. Mascot scoring showed that p > 0.01 was equivalent to a score of 24. The criterion for a positive identification of proteins identified with at least 2 peptides was a minimal score of 24 for each peptide which represents a 1:10,000 false positive rate at protein level. The maximal score for a peptide from a reversed entry of the annotated M. tuberculosis H37Rv database was found to be 31 Buspirone HCl (data not shown). This was considered as a threshold for false-positive identifications, and all proteins identified in this study with only one peptide were based on a score higher than 37 (25:10,000). No false positive identifications were observed from the reversed database using these criteria. For visualization and validation of spectra, MSQuant version +1.4.2 was used. MSQuant is an open source tool available at http://​msquant.​sourceforge.​net and is widely used for LC-MS/MS data analysis [51]. Western blot Proteins from both lipid and aqueous phase were separated by SDS-PAGE, electroblotted to nitrocellulose membranes (Amersham Biosciences) and blocked with 5% non-fat milk in PBS containing 0.5% Tween 20 (PBST) for 1 hour at RT. The membranes were then washed with PBST for 10 min. This was repeated three times.

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