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. 2013 May 15;13(5):602-612.
doi: 10.1016/j.chom.2013.04.008.

The Mtb Proteome Library: A Resource of Assays to Quantify the Complete Proteome of Mycobacterium Tuberculosis

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The Mtb Proteome Library: A Resource of Assays to Quantify the Complete Proteome of Mycobacterium Tuberculosis

Olga T Schubert et al. Cell Host Microbe. .
Free PMC article

Abstract

Research advancing our understanding of Mycobacterium tuberculosis (Mtb) biology and complex host-Mtb interactions requires consistent and precise quantitative measurements of Mtb proteins. We describe the generation and validation of a compendium of assays to quantify 97% of the 4,012 annotated Mtb proteins by the targeted mass spectrometric method selected reaction monitoring (SRM). Furthermore, we estimate the absolute abundance for 55% of all Mtb proteins, revealing a dynamic range within the Mtb proteome of over four orders of magnitude, and identify previously unannotated proteins. As an example of the assay library utility, we monitored the entire Mtb dormancy survival regulon (DosR), which is linked to anaerobic survival and Mtb persistence, and show its dynamic protein-level regulation during hypoxia. In conclusion, we present a publicly available research resource that supports the sensitive, precise, and reproducible quantification of virtually any Mtb protein by a robust and widely accessible mass spectrometric method.

Figures

Figure 1
Figure 1. The Mtb Proteome Library Workflow in Three Phases
(A) Phase I, Proteome Mapping: After harvesting bacterial cultures, proteins are extracted and digested with the proteolytic enzyme trypsin. The resulting peptides are separated into 24 fractions using off-gel isoelectric focusing to reduce sample complexity, and each fraction is analyzed by discovery-driven MS. Peptide identifications can then be used to infer proteins that were present in the sample. The peptide and protein identifications, as well as the corresponding spectra, can be browsed interactively in the PeptideAtlas database (http://www.PeptideAtlas.org). (B) Phase II, Proteome Library Generation: From the collected data, the most MS-suited, unique peptides are selected for every annotated protein of Mtb. For proteins that have never been observed previously, representative peptides are predicted. The peptides are synthesized, pooled in mixes of 96, and analyzed in SRM-triggered MS2 mode (SRM-MS2). From the resulting spectra the most intense fragment ions, as well as the chromatographic retention times, can be extracted. These mass spectrometric coordinates, called SRM assays, constitute the synthetic Mtb Proteome Library and can be downloaded from the SRMAtlas database (http://www.SRMAtlas.org). (C) Phase III, Proteome Library Validation: The SRM assays in the synthetic Mtb Proteome Library are validated for the detection of proteins in unfractionated mycobacterial lysates by SRM. The resulting quantitative SRM traces and statistical scores can be viewed in the PASSEL database (http://www.PeptideAtlas.org/passel).
Figure 2
Figure 2. Defining the MS-Accessible Proteome of Mtb by Discovery MS
(A) Proteome coverage and distribution of the peptides detected by discovery MS in an extensively fractionated lysate of Mtb during exponential and stationary growth. (B) Proteome saturation analysis showing the progression of protein identifications as a function of identified spectra. The red line represents data acquired from the fractionated Mtb lysate; the black and blue lines represent the simulated progressions of all identifications and the true positive identifications at an FDR of 1%. The vertical gray line marks the size of our data set in terms of acquired peptide-spectrum matches. One hundred percent proteome saturation is defined at 300,000 peptide-spectrum matches. (C) Comparison showing the overlap of the protein identifications with the transcriptome determined by RNA sequencing (Arnvig et al., 2011). The numbers represent the total identifications in exponential and stationary phase cultures for both technologies.
Figure 3
Figure 3. Generation and Validation of the Mtb Proteome Library
(A) Proteome coverage and distribution of the 15,679 synthetic peptides for which a fragment ion spectrum, and thus an SRM assay, could be obtained. (B) Proteome coverage and distribution of the 7,094 peptides for which the synthetic SRM assay could be validated by SRM in a mixed unfractionated Mtb lysate of exponential and stationary phase cultures. (C) Theoretical specificity of SRM assays determined by the SRMCollider algorithm is shown as a cumulative plot of the number of peptides which can be uniquely identified with a given number of transitions. Transitions were selected with decreasing intensity. Scheduled indicates that only background peptides with a retention time close to the target peptide are taken into consideration as interfering background. See also Figures S1 and S2 and Table S1.
Figure 4
Figure 4. Proteome-wide Absolute Abundance Estimates for Mtb
SRM-based absolute label-free abundance estimates for every protein identified by SRM with two or more peptides (2,195 proteins). The abundance estimate has a mean fold error of 2.1 ± 0.6. (A) Abundance distribution of all quantified proteins. (B) Absolute concentrations of the ten most abundant proteins in Mtb. (C) Absolute concentrations mapped on selected protein classes and the metabolic network of Mtb (http://pathways.embl.de/iTuby). Colors correspond to the ones in (A). (D) Abundance distribution among the functional classes of Mtb as defined in TubercuList. The first and second rows show the distribution of genes in the genome and of the quantifiable proteins, respectively. The third row shows the relative protein concentration for each functional class. Abbreviations are as follows: Met, metabolism; TCA, tricarboxylic acid cycle; CH, carbohydrate; AA, amino acid; and Vit, vitamine and cofactor. See also Figure S3 and Table S2.
Figure 5
Figure 5. Time-Resolved Protein Level Regulation of the DosR Regulon under Hypoxic Stress
(A) Growth curve of three exponentially growing M. bovis BCG cultures subjected to hypoxic conditions in a standing culture model. Agitation of shake-flask cultures was stopped at day 0 and resumed after 6 days (reaeration). (A)–(C) represent biological replicates. (B) Summary table of the DosR regulon study. (C and D) Fold changes versus day 0 as determined by SRM using heavy isotope-labeled reference peptides for each protein. The data were subjected to hierarchical clustering, and each of the ten clusters was colored differently (corresponding colors in C and D). (E) For two operons of the DosR regulon the protein fold changes after 4 days of exposure to hypoxia are shown. Error bars represent the standard error. See also Figures S4 and S5 and Table S3.
Figure 6
Figure 6. Proteogenomics Combined with the Mtb Proteome Library Reveals Previously Unannotated Proteins
(A) and (B) exemplify two peptides that belong to a protein, identified by proteogenomics, which has so far not been annotated in Mtb H37Rv. Spectra were acquired from synthetic peptides, whereas SRM traces were acquired from endogenous peptides in a whole-cell lysate. See also Table S4.

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