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. 2010 May;10(10):1966-84.
doi: 10.1002/pmic.200900836.

Descriptive proteomic analysis shows protein variability between closely related clinical isolates of Mycobacterium tuberculosis

Carolina Mehaffy  1 , Ann Hess, Jessica E Prenni, Barun Mathema, Barry Kreiswirth, Karen M Dobos
Affiliations

Descriptive proteomic analysis shows protein variability between closely related clinical isolates of Mycobacterium tuberculosis

Carolina Mehaffy et al. Proteomics. 2010 May.

Abstract

The use of isobaric tags such as iTRAQ allows the relative and absolute quantification of hundreds of proteins in a single experiment for up to eight different samples. More classical techniques such as 2-DE can offer a complimentary approach for the analysis of complex protein samples. In this study, the proteomes of secreted and cytosolic proteins of genetically closely related strains of Mycobacterium tuberculosis were analyzed. Analysis of 2-D gels afforded 28 spots with variations in protein abundance between strains. These were identified by MS/MS. Meanwhile, a rigorous statistical analysis of iTRAQ data allowed the identification and quantification of 101 and 137 proteins in the secreted and cytosolic fractions, respectively. Interestingly, several differences in protein levels were observed between the closely related strains BE, C28 and H6. Seven proteins related to cell wall and cell processes were more abundant in BE, while enzymes related to metabolic pathways (GltA2, SucC, Gnd1, Eno) presented lower levels in the BE strain. Proteins involved in iron and sulfur acquisition (BfrB, ViuB, TB15.3 and SseC2) were more abundant in C28 and H6. In general, iTRAQ afforded rapid identification of fine differences between protein levels such as those presented between closely related strains. This provides a platform from which the relevance of these differences can be assessed further using complimentary proteomic and biological modeling methods.

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Conflict of interest statement

All of the authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Representative 2D gels from the secreted fraction of each analyzed strain (Clockwise: CDC1551, BE, H6, C28). Numbers correspond to proteins in Table 1. pH range is 4 – 7.
Figure 2
Figure 2
Differential CFP (Panel A) and Cytosolic (Panel B) proteins between BE and both, C28 and H6 (p-value <0.05) obtained by iTRAQ labeling followed by MS/MS analysis. Note: PrcB, Rv0569 and Rv2493 abundance values were also significantly different between BE and CDC1551 in the CFP. All cytosolic proteins, with exception of Rv2302 and Rv0020c also presented significantly different abundance values between BE and CDC1551.
Figure 3
Figure 3
Relative abundance of proteins identified in the CFP (secreted) or cytosolic fraction by iTRAQ labeling followed by MS/MS analysis. NSAF: Normalized Spectral-counts Abundance Factor. Note: Spectral counts were compiled from both biological and technical replicates.
Figure 4
Figure 4
Comparison of Cfp2 levels between A: 2D-GE and B: iTRAQ. Notes: Please refer to Figure 1 and Table 1 for spot identification. Note that several peptides were used for identification and quantification of Cfp2 by iTRAQ. Only two are shown in this figure.
See this image and copyright information in PMC

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