Abstract
Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.
Highlights
According to the World Health Organization (WHO), tuberculosis (TB) ranks as the second leading cause of death from an infectious disease worldwide, after HIV (WHO|Global tuberculosis report 2014, 2014)
In this study we set out to analyse the phosphoproteome of a hyper-virulent Beijing genotype M. tuberculosis isolate by extracting whole cell lysate proteins at early-logarithmic growth (OD600 of 0.8) (Figure 1) which resulted in the identification of 619 MS/MS spectra
The 274 LC-MS/MS spectra that fulfilled the criteria for high confidence phosphosites identified a total of 414 (38:59:3%) S/T/Y phosphorylation sites present in 214 M. tuberculosis H37Rv proteins (Supplementary Table S2; Supplementary Figure S1)
Summary
According to the World Health Organization (WHO), tuberculosis (TB) ranks as the second leading cause of death from an infectious disease worldwide, after HIV (WHO|Global tuberculosis report 2014, 2014). Of particular interest is the identification of proteins with post-translational modifications (PTMs) as these modifications are often critical to protein functions, such as regulating protein-protein interactions, subcellular localization or modification of catalytic sites (Seo and Lee, 2004; Gupta et al, 2007). Protein phosphorylation is an important reversible PTM that directly or indirectly regulates signal transduction cascades linking the intracellular and extracellular environments. Protein phosphorylation plays a fundamental role in the regulation of key processes ranging from metabolism and cellular homeostasis to cellular signaling which can be mediated by two classes of phosphorylation events (Cozzone, 1998). The underlying molecular mechanisms regulating protein phosphorylation and dephosphorylation is of great physiological importance due to its ability to www.frontiersin.org
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