| The rapid development of mass spectrometry-based proteomic technologies, and the increased accessibility of powerful data analysis tools, have provided proteomic researchers the ability to look at changes beyond only qualitative measurements and into the quantitative intricacies of biological systems. SILAC, or Stable Isotope labeling by Amino acids in Cell culture, is the one of such technologies that has been applied effectively and provided highly reproducible and accurate quantitative measurements. SILAC’s initial applications were limited to yeast and cultured cells but more recently has been applied to fruit flies and mice. The technology has not been readily applied to such organism as Escherichia coli because of the lackness of the availability of SILAC medium. Here we present a mass spectrometry-based quantitative proteomics method to first introduce SILAC technology into E. coli via development of a novel growth medium and secondly to examine global proteomic profile changes during IPTG inducing.E. coli is a rod-shaped gram-negative bacteria that has become one of the most useful and characterized models because of its ease in culture and manipulation. Its genome has been completely sequenced and its proteome has also been well annotated. While E. coli has been widely used both in industry and laboratory research, it has rarely used in SILAC based methods because of a lack of proper labeling medium. Complete labeling is essential to the success of SILAC and the proper medium is an important prerequisite. We have developed a medium with a base composition of M9minimal growth medium and yeast synthetic complete (SC) medium. By adjusting the composition of various amino acids and concentrations of carbon sources, we were able to create a medium that allowed fast growth and complete labeling within just10hours.To test the accuracy of our quantitative platform we performed a pure null experiment involving both light and heavy labeling of BL21(DE3) competent cells in our SILAC medium. The two cellular populations were mixed at a ratio of1:1according to OD600and quantitative proteomics was performed. The quantitative results showed us the accuracy of our platform is about7%which was the highest so far based on our published literature. In addition, the pure null experiment also allowed us to figure out the necessary signal cutoff and filter thresholds for our following experiments. Induction by IPTG is commonly used to differentially express proteins in E. coli. During induction, the cells are driven to mass transcribe objective gene of interest. We induced an exogenous protein by IPTG for1hour in BL21(DE3) recombinant cells, and collected the cells before and after induction which were differentially cultured in light and heavy medium respectively. After LC-MS/MS analysis we filtered the data using the cutoffs and thresholds from our pure null experiment and summarized the results according to differential expression. SILAC results showed that most proteins were down-regulated. These proteins were but not limited to tRNA synthesis, tricarboxylic acid cycle (TCA), ribosomal function, and flagella function. There were11proteins that showed over-expression profiles,3of which are expected as they were T7RNA polymerase, LacZ, and the target protein. The remaining over-expressed proteins included3related to cell wall synthesis suggesting that cellular morphology is changed. This was validated by the observation with electro-microscope. This experiment shows that under such conditions as induction the cellular state is changed and the media we developed can be as a good tool for quantitative proteomics research. |
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