The reproducibility of chromatographic separation and signal intensities for the twelve 5-h runs was excellent, as assessed from data for selected tryptic peptides identified in the bacterial lysate preparation. Variations in retention time for the selected peptides selleck screening library were in the range of 0.32-1.05%, and variations for precursor ion current AUCs were in the range of 5-14% over the 3
day period. This high level of reproducibility can be attributed to two factors: (i) the highly reproducible chromatographic configuration described above, and (ii) the efficient precipitation/on-pellet-digestion procedure that removed detergents and other potentially interfering compounds. Current methods for proteomic investigation are prone to false-positives arising from technical variability [34].In this study, to eliminate false-positives resulting from drift in nano-LC or ionization efficiency, for example, and possible instability
of certain tryptic peptides, all samples were analyzed in a random order.To evaluate the false-positive rate before comparing the bacterial samples AZD1480 price grown under different conditions, we designed an experiment to determine the false-positive rate in relative quantification. From the 10 repetitive analyses of a find more pooled bacterial sample (above), 5 runs were randomly assigned as the control group, and the remaining 5 were designated as the experimental group. Expression profiles between the two groups were then compared. In total, 32,178 ion-current frames were matched among the two groups of samples using Sieve. The observed distribution of peptide ratios (experimental:control) concentrated narrowly around 1.0, with
96% of ion-current frames in the range of 0.9-1.1. Approx. 1% of ions differed by more than 15% of the 1.0. Only 2 peptides were identified as significantly selleck chemicals llc changed between the two groups at p < 0.05.Such a low false-positive rate and high quantitative precision supported the suitability of this method for profiling of the bacterial samples using the replicate number (n = 5) selected. Proteomic profiling of H. influenzae grown in chemically defined media with and without sputum Previous analyses of the H. influenzae proteome have employed electrophoresis-based studies [35–40] to identify abundantly expressed proteins under laboratory growth conditions.More recently Kolker et al [41] employed a direct proteomics approach using liquid chromatography with ion trap tandem mass spectroscopy and identified 414 protein with high confidence, including 15 proteins that were encoded by genes that were previously annotated as conserved hypothetical proteins.
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