Comparison of predicted and observed properties of proteins encoded in the genome of Mycobacterium tuberculosis H37Rv

Abstract

Proteome studies complement current molecular approaches through analysis of the actively translated portion of the genome (the "functional proteome"). Two-dimensional gel electrophoresis (2-DGE) utilising immobilized pH gradients of pH 2.3-5.0 and pH 6.0-11.0, developed with predetermined regions of overlap compatible with commercially available pH 4.0-7.0 gradients, permitted the display of a significant portion of the proteome of Mycobacterium tuberculosis H37Rv. A significant portion of the M. tuberculosis proteome, in the molecular mass (M(r)) window 5 kDa to 200 kDa and with isoelectric point (pI) between pH 2.3 and 11.0, was visualised for the first time. A total of 493 protein spots were effectively resolved, including 126 spots that could not be seen using standard pH 4.0-7.0 gradients. These results were used to compare the physical properties of the observed proteins to the theoretical predictions of the recently completed M. tuberculosis H37Rv genome. Most proteins were found in the pI and mass window of pH 4.0-7.0 and 10-100 kDa. Analysis of the predicted proteome revealed a bimodal pI distribution, with substantial numbers of proteins in the pI regions 4.0-7.0 and 9.0-12.0 as has been seen for the majority of completed genomes. Such data may reveal current limitations in experimental extraction and separation of extremely basic, high M(r) and hydrophobic proteins via 2-DGE. Conversely, 13 acidic proteins were observed with pI less than the lowest value predicted by the genome. In addition, a subset of small protein (< 10 kDa) were observed within the pI region of pH 5.0-8.0 that were not predicted by the complete genomic sequence, reflecting the current inability to distinguish small genes from within DNA sequence. This work represents the foundation for comparing the protein expression patterns of different pathogenic and nonpathogenic M. tuberculosis strains. The characterization of M. tuberculosis protein expression, further facilitated by the recent completion of the genome sequence, could aid in developing more effective diagnostic or therapeutic reagents.