Determination of global protein turnover in stressed mycobacterium cells using hybrid-linear ion trap-Fourier transform mass spectrometry

作者：Rao Prahlad K; Roxas Bryan A P; Li Qingbo^*

来源：Analytical Chemistry, 2008, 80(2): 396-406.

DOI：10.1021/ac701690d

We determined the global protein turnover profiles for Mycobacterium smegmatis under acid shock and iron starvation conditions using a simple N-15 isotope doping technique and a complete medium replacement method for chasing. We used a high-resolution hybrid-linear ion trap-Fourier transform mass spectrometer coupled with nanoliquid chromatography separation to measure protein turnover values for 151 proteins over a dynamic range of 3 orders of magnitude ranging from about 0.2 to 500. Of these 151 proteins, 31 had significant protein turnover changes (p < 0.65) at both stress conditions and had protein turnover values increased or decreased by more than 2-fold under at least one stress condition. Protein turnover increased under acid shock for 28 of the 31 proteins but decreased under iron starvation for all the 31 proteins. Only two proteins had protein turnover lowered by more than 2-fold (p < 0.05) under both stress conditions, including an ATP synthase F1 beta subunit (MSMEG4921; AtpD) and a catalase/peroxidase (MS-MEG6346; KatG). KatG is required for in vivo activation of isoniazid to be bacterialcidal. Decrease of KatG protein turnover under both stress conditions supports the view that isoniazid may induce a dormancy program in mycobacteria, which in turn limits the efficacy of this drug against dormant subpopulation of mycobacteria. Thus, measuring protein turnover in stressed Mycobacterium cells has implications in understanding drug action and resistance mechanisms.

• 出版日期2008-1-15