This protocol uses refractive index detection to measure byproducts of carbon metabolism that are commonly accumulated in lysate-based, cell-free systems. It also makes use of mass spectrometry to detect an even broader panel of central metabolites that are generated in metabolically active lysates. The two techniques used in this protocol provide the ability to quantitatively describe the chemical reactions occurring in a lysate-based, cell-free system, making it possible to detect a wider range of metabolites, including those present at low concentrations in the complex lysate background.
Demonstrating the procedures will be Jaime Lorenzo Dinglasan and David Reeves, graduate researchers from the Biosciences Division. Begin by combining the different components in 1.5-milliliter microcentrifuge tubes to prepare final reactions with 4.5 milligrams per milliliter of total lysate protein. Prepare cell-free metabolic engineering, or CFME, reactions with final volumes of 50 microliters in triplicate per time point.
Terminate the triplicate reactions at their appropriate time points immediately by adding an equal volume of 5%trichloroacetic acid to each sample's final reaction volume. Then dilute each sample by adding two times the volume of sterile water to each reaction mixture. To recapitulate time zero, mix the same volume of 5%trichloroacetic acid as the total final reaction volume with the lysate prior to adding the rest of the reaction components.
This acidification step precipitates lysate enzymes before they significantly metabolize glucose. Vortex the samples and centrifuge on a benchtop microcentrifuge at 11, 600 times g for five minutes, and transfer the supernatants containing the organic analytes to clean tubes. Store the samples at minus 20 degrees Celsius if HPLC analyses are to be conducted later.
Ensure to thaw the stored samples on ice before proceeding to the next step. Filter each supernatant with a 0.22-micrometer pore filter. After centrifuge, transfer each filtrate to a clean HPLC glass vial.
Load the vials onto the autosampler tray of the HPLC system that has already been set up for analysis. From the menu bar, select Sequence, New Sequence Template. Select Sequence.
Save sequence template as sequence template name S.Select Sequence, Sequence Table. Append N rows corresponding to N vials. Then input vial positions and sample names under Vial and Sample Name, respectively, according to their arrangement on the autosampler tray.
Select the method generated as described in the manuscript from the Method Name dropdown menu, and input 50 microliters as injection per vial for each row. Click Apply, and save the sequence template by selecting Sequence, Save Sequence Template. Ensure the sequence template is loaded by selecting Sequence, Load Sequence Template, sequence template name S.After achieving a stable baseline on the online plot, right-click the panel RID Module, Control, Off Recycling Valve to direct the solvent flow through the RID detector to waste.
To start data acquisition, select Sequence from the menu bar, and then select Sequence Table, Run. Select Data Analysis view from the View menu. Locate the sequence file name from the file list on the left-hand side of the screen.
On the center panel on the screen, go to the Signal View Selection, RID Signal to view the sample chromatograms. Select a row corresponding to any of the samples from the top panel on the screen. Peaks corresponding to the target analytes will be arranged along the retention time axis as glucose, succinate, lactate, formate, acetate, and ethanol in samples where all these metabolites are present.
Discern whether the peaks of interest are well integrated by the software. A red line should be automatically drawn as the base of each peak. If the red line is askew, the automatic integration has failed.
Then select the Manual Integration button from the Integration Tool Set, and manually draw a peak base to integrate the peak area. Select the Cursor tool from the Common Tool Set to click on properly integrated peaks. The peak area and the corresponding reaction time of the selected peak will be highlighted as a table row on the bottom panel off the screen.
To export peak areas, select File, Export, Integration Results. Plot peak area values versus known concentrations of samples in a spreadsheet. Right-click on the plotted data, and then select Add Trendline, Format Trendline, Display Equation on Chart.
In a separate spreadsheet, use the equations of standard curve trendlines to convert peak area values to concentrations for every analyte from each sample. Calculate the average peak areas and standard error values across triplicates for data visualization. Set up triplicate reactions per time point, except time zero, on ice as described in the manuscript.
Instead of glucose, use a final concentration of 100-millimolar glucose-13C6 in the reactions. Incubate the reactions at 37 degrees Celsius for one, two, and three hours. To begin with the analysis, pipette an equivalent volume of the extraction solvent to each sample.
If the samples were frozen, add the extraction solvent before the samples completely thaw out to prevent the reactivation of glucose metabolism. Perform all sample processing steps on ice. To recapitulate time zero, pipette the final volume of extraction solvent to an appropriate volume of lysate for the desired final concentration of 4.5 milligrams per milliliter in 50 microliters of reaction volume.
Add the rest of the reaction components as described previously. This acidification step precipitates lysate enzymes before they significantly metabolize glucose. Incubate the samples in extraction solvent on ice for 30 minutes with gentle shaking.
Then centrifuge the samples at 21, 000 times g for 15 minutes at four degrees Celsius to separate the supernatant from the precipitated protein. Transfer 50 microliters of the supernatant to autosampler vials, and load the vials onto the tray within the four degrees Celsius autosampler. After preparing the instrument for analysis, set up a run sequence using the LC-MS/MS system's data acquisition and interpretation software.
Within Roadmap, Sequence Setup, right-click on the table to insert as many rows as samples. For each row, set the injection volume to five microliters and the position to the vial's respective position on the autosampler tray. Input file names as sample names, and set the desired file path for run results.
To start the run, highlight all file names in the sequence. From the menu bar, select Actions, Run Sequence. Open MZmine, and import the raw output files obtained previously.
From the menu bar, select Raw Data Methods, Raw Data Import, and select the files corresponding to the samples. Follow the steps described in the manuscript to complete the MZmine analysis. Export the raw peak areas at the end of MZmine analysis as a CSV file.
Open a spreadsheet to calculate the masses of 13C-labeled metabolites from glucose metabolism for the targeted search. Use calculated masses of 13C-labeled metabolites to search and annotate mass-to-charge features from MZmine results. Manually check the spectra of the putative annotations on a quality browser to confirm the annotations.
Open Roadmap, Qual Browser. From the tool bar, open raw file to import raw MS data of each sample. Draw a line under the desired range of retention times corresponding to the putative annotation on the total ion chromatogram to view a mass spectrum.
In HPLC-RID experiments, glucose was consumed within the first three hours of the reaction and mainly fermented to lactate. Ethanol accumulation also significantly occurred within the first three hours of the reaction and stopped thereafter. Acetate was initially present in the reactions as a component of the S30 buffer and only accumulated due to metabolism after six hours, when glucose consumption had slowed down.
Lactate and ethanol can thus be considered as the major fermentation end-products in lysate-based, cell-free glucose metabolism. It was observed that both formate and succinate were synthesized as minor fermentation products. Glucose-13C6 was observably consumed through glycolysis, as evident by the fluctuations in glycolytic intermediates.
Consistent with the HPLC refractive index detection data, glucose accumulated to lactate-13C3 and was also fermented to succinate-13C3 within the first three hours of the reaction. The incorporation of glucose-13C6-derived carbons to sugar phosphates 6-phosphogluconolactone, 6-phosphogluconate, ribulose-5-phosphate, and sedoheptulose-7-phosphate was also observed, confirming the participation of the pentose phosphate pathway in lysate glucose metabolism. The lysate glucose metabolism was found to feed tyrosine-13C9 synthesis, while also providing a precursor for histidine-13C5 production.
It is important that the control samples precisely represent time zero. Therefore, ensure that the proteins in the lysate are inactivated prior to mixing in the solution containing energy sources. Also ensure that the automatic integration of peaks from test samples, control samples, and standards is consistent as well, so that extracted peak areas are comparable.
