Cell-free protein synthesis is an emerging technology in systems and synthetic biology for the in vitro production of proteins. Cell-free protein synthesis systems have no cell wall, thus we have direct access to metabolites. In this protocol, we quantify the absolute measurements of 40 metabolites involved in central carbon and energy metabolism, which allows us to characterize cell-free metabolism.
The protocol relies on the metastasizing the compounds with aniline, which allows for better separation and higher mass spectrometry resolution. In addition, we use internal standards with an isotopic we label aniline, for absolute quantification of the metabolites. The tagged metabolites coelute, which eliminates the effects of iron suppression.
Allowing for the accurate quantification of the compounds. Working in a hood, combine 550 microliters of the aniline, with 337.5 microliters of LC-MS grade water. And 112.5 microliters of 12 molar hydrochloric acid in a centrifuge tube.
Vortex well, and store the six molar aniline solution at pH 4.5 and four degrees Celsius. To prepare a six molar carbon-13 aniline solution, at pH 4.5, combine 250 milligrams of carbon-13 aniline with 132 microliters of water, and 44 microliters of 12 molar hydrochloric acid. Vortex well, and store at four degrees Celsius.
To prepare 200 milligrams per milliliter EDC solution, dissolve two milligrams of EDC in 10 microliters of water for every sample to be tagged. And vortex well. To prepare samples, quench and precipitate the proteins in a cell-free protein synthesis reaction, by adding an equal volume of ice-cold 100%ethanol to the cell-free synthesis reaction.
Centrifuge the sample at 12, 000 times g, for 15 minutes at four degrees Celsius. Transfer the supernatant of the sample to a new centrifuge tube. To label the sample with carbon-12 aniline solution, transfer six microliters of the sample into a new centrifuge tube, and bring the volume to 50 microliters with water.
Add 5 microliters of 200 milligrams per milliliter EDC solution, mix the carbon-12 aniline solution well, and transfer 5 microliters into the tube. Vortex the reaction with gentle shaking for two hours at room temperature. After two hours, remove the tubes from the shaker and transfer them into a fume hood.
Add 1.5 microliters of triethylamine to each reaction to stop the aniline tagging reaction and stabilize the compounds. Centrifuge at 13, 500 times g for three minutes. And save the supernatant.
To label the internal standards with carbon-13 aniline solution, dilute the internal stock solution to 80 micromolar with a final volume of 50 microliters. Add five microliters of the 200 milligrams per milliliter EDC solution, and five microliters of the well-mixed carbon-13 aniline solution. Vortex the reaction with gentle shaking for two hours at room temperature.
After two hours, remove the tubes from the shaker, and add 1.5 microliters of triethylamine to the reaction in a fume hood. Centrifuge at 13, 500 times g for three minutes and save the supernatant. To divine the tagged internal standard, and the tagged sample, mix 25 microliters of each in an autosampler vial.
And analyze by the LC-MS procedure. Then, to create a standard curve for untagged metabolites, first, dilute the stock solution of untagged metabolites to different concentrations, with volume of 50 microliters. Add five microliters of the 200 milligrams per milliliter EDC solution, and five microliters of carbon-12 aniline solution to each concentration.
Vortex the reaction with gentle shaking for two hours at room temperature. And proceed with the triethylamine treatment. And centrifugalization before analyzing by the LC-MS as previously.
After initializing LC-MS, according to manufacturer's instructions, inject five microliters of the sample into the column. And acquire the appropriate m over z ion intensities for the carbon-12 aniline tagged sample. Then, inject five microliters of the same sample again.
And acquire the m over z ion intensities for the carbon-13 aniline tagged standards. In this protocol, metabolites expressing green fluorescent protein in an E.coli-based cell-free protein synthesis were quantified. 40 metabolites involved in central carbon and energy metabolism, were detected and quantified using internal standards.
While a standard curve for five of the metabolites that were not tagged with aniline was also developed. The diverse metabolites involved in these pathways were a class of phosphorylated sugars, phospho-carboxylic acids, carboxylic acids, nucleotides, and co-factors. In addition, the method enabled the separation of structural isomer pairs, such as glucose-six phosphate, and fructose-six phosphate in a single LC-MS run.
The most important step is labeling the de-proteinized sample with aniline. Since the aniline solution separates, it is important to work quickly and effectively, while maintaining good safety practices. This method helps to characterize cell-free metabolism with the quantification of 40 metabolites.
But, additional compounds are in the cell-free mixture, such as amino acids that can be quantified. This would help in providing a more comprehensive look into cell-free metabolism. This method revealed that cell-free systems have complex metabolisms, still operating that can be potentially exploited for better energy efficiency and carbon yield.
The protocol relies on tagging the compounds with aniline with the use of EDC as a catalyst. Both these reagents are hazardous and should be used with caution.
