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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 H(37)Rv, 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.
