Proteogenomic Analysis of Mycobacterium tuberculosis By High Resolution Mass Spectrometry

Abstract

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ϳ80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ϳ250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

“…We immunoprecipitated HOAS from M. tuberculosis H37Rv lysates using ␣-HOAS antibodies (7) and subjected the band corresponding to the target protein to Edman sequencing. The first 5 amino acids were determined to be ANISS in agreement with recent observations (15). Next, HOAS was cloned into the pET-11c vector, and tagless recombinant HOAS was overexpressed and purified to Ͼ90% purity as judged by SDS-PAGE.…”

Disclosure of Selective Advantages in the “modern” Sublineage of the Mycobacterium tuberculosis Beijing Genotype Family by Quantitative Proteomics

“…This combined information is a strong indication that Rv2688c is more abundant in M. tuberculosis Beijing strains than in the proteome of M. tuberculosis H37Rv. Rv3728 was reported to be present in the proteome of M. tuberculosis H37Rv as well (74). Howbeit, peptides corresponding to Rv3728 were only identified by the Sequest algorithm, whereas Mascot and MassWiz failed to identify any peptide belonging to Rv3728.…”

“…Three efflux pumps, Rv0341/iniB, Rv2688c, and Rv3728, were exclusively identified in our dataset on Beijing strains. However, a proteogenomic approach that studied the proteome of M. tuberculosis H37Rv identified the respective proteins, Rv0341/iniB, Rv2688c, and Rv3728 (74). It should be noted that 123 LC-MS/MS analyses were performed, which resulted in the identification of Rv2688c by a single peptide.…”

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

“…Most of the proteomic studies to date have focused on the bacilli grown under normoxia (22,23), or during transition from normal replicating stage to dormancy (24). Starck et al used 2-D electrophoresis to compare the proteomes of MTB grown under aerated and anerobic conditions, and found 50 proteins differentially expressed under the latter (12).…”