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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