Proteomic analysis of mycobacteria and mammalian cells

Abstract

Tuberculosis is a serious threat that claims 2 million lives annually. Mycobacterium tuberculosis is the causative agent of tuberculosis. The fast-growing bacteria Mycobacterium smegmatis is a model mycobacterial system, a non-pathogenic soil bacterium that nonetheless shares many features with the pathogenic M. tuberculosis. Multidimensional chromatography coupled with shotgun style tandem mass spectrometry was used to detect and identify 2,550 distinct proteins from M. smegmatis with an estimated 5% false positive identification rate, many predicted genes were annotated using experimental results and protein expression levels were estimated from the shotgun proteomic data. First, in 25 exponential and stationary phase experiments, we observed numerous proteins involved in energy production, protein translation, and lipid biosynthesis. Protein expression levels were estimated from the number of observations of each protein, allowing measurement of differential expression of complete operons, and the comparison of the stationary and exponential phase proteomes. Expression levels are correlated with proteins' codon biases and mRNA expression levels. Secondly, we measured changes in the proteome of Mycobacterium smegmatis in response to three anti-tuberculosis drugs isoniazid (INH), ethambutol (EMB) and 5- chloro-pyrazinamide (5-Cl-PZA). Protein expression levels were calculated from the number of identified peptides for each protein. Translation, energy production, and protein export are all down-regulated in the three drug treatments. By contrast, systems related to drugs’ targets, including lipid, amino acid, nucleotide metabolism and transport, show specific protein expression changes associated with each drug treatment. We use these changes to infer likely targets for PZA. Thirdly, computational methods were used to predict protein-protein interactions and protein functions in a metabolic pathway in M. tuberculosis. Protein functional links were built and specific functions were characterized for the pathway and its parallel pathways in M. tuberculosis and other organisms. Finally, multidimensional chromatography coupled with shotgun style tandem mass spectrometry has been applied in the analysis of nuclear proteins from mammalian cells. Nuclear proteins were identified from mouse T lymphoma cells. Nuclear matrixassociated proteins were identified from human preliminary T cells during the transition from quiescent state to proliferating state. These proteins are involved in the function of DNA replication, RNA transcription, splicing, etc.