A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

The overall goal of this procedure is to study metabolic profiling in cultured cells. This is accomplished by first extracting polar metabolites from cells. The second step is to reconstitute the cell extracts in water and transfer the samples to lc vials.

Next, the samples are injected into the lc QE MS instrument, which has been calibrated and raw data is recorded in the computer in the final step. Metabolite peaks are extracted from the raw data using commercial software, and unknown peaks are searched against a high resolution mass database. Though this master can provide insight into relative metabolite levels in sars, it can also be applied to other systems such as serum or tissues.

First, prepare 500 milliliters of mobile phase A, which is 20 millimolar ammonium acetate, and 15 millimolar ammonium hydroxide in 3%aceto, nitrile, and water. After loosely capping the bottle, sonicate the solution in a water bath sonicate for 10 minutes without heating. Following this dissolve five milligrams of both sodium fluoro acetate, and acid in five milliliters of water to make a final concentration of one milligram per milliliter.

Then dissolve diazinon in methanol to make a final concentration of 10 micrograms per milliliter. To prepare one milliliter of negative low mass calibration solution, mix 960 microliters of thermo negative calibration solution with 20 microliters of the sodium fluoro acetate and homovanillic acid solution. To make one milliliter of positive low mass calibration solution mix 990 microliters of thermo positive calibration solution and 10 microliters of the diazinon solution.

After carrying out a standard mass calibration in positive mode, adjust the scan range to 60 to 900 mass to charge ratio in the instrument control panel and apply a source collision induced dissociation of 25 electron volts. Input customized calibration ions, and once the ion source is stable, start the customized calibration in the tuning page. Switch the polarity to negative mode after carrying out a standard mass calibration in negative mode, adjust the scan range to 60 to 900 mass to charge ratio in the instrument control panel and apply a source collision induced dissociation of 35 electron volts.

Input customized calibration ions, and once the ion source is stable, start the customized calibration. In the tuning page, equip the QE MS instrument with the heated electro spray ionization or HESI probe by fixing it at level C and then connecting it to the nitrogen gas inlets, vaporizer cable, and voltage cables. In the computer tuning page, set the relevant tuning parameters for the probe.

Set the capillary temperature at 320 degrees Celsius and the S lens at 55. Next in the method program, build a full scan method using the following values. Establish the chromatography method by inputting the linear gradient information.

Employ an amid column for compound separation at room temperature. At this point, prepare an extraction solvent by manually mixing 40 milliliters of methanol and 10 milliliters of water in a 50 milliliter.Two. When previously cultured colon cancer H CT eight cells reach 80%confluence.

Quickly aspirate the medium and place the six well plate on top of dry ice. Then immediately add one milliliter of extraction solvent to each well and transfer the plate to a negative 80 degrees Celsius freezer. After removing the plate from the freezer, 15 minutes later, place it on top of dry ice and scrape the cells into the solvent.

Transfer the solution from each well to three 1.7 milliliter einor tubes and centrifuge the samples with a speed of 20, 000 times G at four degrees Celsius for 10 minutes. Following centrifugation, transfer the supernatant to two new einor tubes. Then dry the samples in a speed vacuum.

Once the samples are dry, store them in the negative 80 degrees Celsius freezer. After removing the samples from the freezer for analysis, allow them to reach ice temperature. Then add 20 microliters of ice cold water, and vortex the samples to dissolve metabolites.

Next, centrifuge the samples at 20, 000 times G at four degrees Celsius. For two minutes, transfer the SUPERNAT to lc vials and inject five microliters of the samples into the lc QES instrument for analysis. Once the calibration has been properly carried out on the QE MS instrument, equilibrate the lc column for five minutes with 85%of mobile phase B at a flow rate of 0.15 milliliters per minute.

Next, set up the sample sequence in a random order to distribute the fluctuations introduced by the LCMS to each sample and ensure a more accurate comparison between different samples. After every six samples, add a wash run, followed by a blank sample to assess the system background and carryover levels. Save the sequence and start the sequence run.

Once the lc column shows stable pressure close to 400 PSI, after the first two samples of the sequence have run, check the peaks in these chromatograms for unknown metabolites to ensure the sample sequence runs smoothly. After all of the samples in the sequence are finished, perform data analysis on a separate computer. Choose the method of peak alignment and frame extraction for small molecule on commercially available software.

Then load the lc ms raw data and group them based on sample types. Pick samples in the middle of the run sequence as a chromatography reference sample for peak alignment, and pick a group as the ratio group or the full change calculation. Then upload a frame seed, including known metabolites for the targeted metabolite analysis with data collected and the corresponding frame seed.

Next, choose full spectra scan either in the positive or negative mode and save this data processing file in the same folder as the raw data. Turn off the database search function and run the workflow. Then export the process data as an Excel sheet containing the peak area of every frame.

For an untargeted metabolite analysis, choose the method of component extraction. Load the sample raw data and three blank samples for background subtraction. Following this group, the raw data and set the reference samples as described previously.

Set the component threshold to 10 to the fifth. Use a human metabolome database for unknown compound identification. After saving this processing file, turn off the database search function and start the workflow.

Once the data processing is finished, use coefficients of variation or CV filters to remove components with large CV within replicate samples and intensity filters. To remove components detected at noise level manually go through each component and pick those with a well-defined peak or relatively big difference in different sample types for the database search. Finally, export the data with hits in the database.

The accuracy of metabolomics data highly depends on the lc QEMS instrument performance to assess whether the instrument is operating in good condition and whether the method applied is proper. Several known metabolite lc peaks are extracted from the total ion chromatography as shown here. Polar metabolites including amino acids, glycolysis, intermediates, TCA, intermediates nucleotides vitamins A TP and NADP have good retention on the column and good peak shapes under the current lc conditions.

Meanwhile, a mass error test is done within 24 hours after low mass calibration as illustrated. Here, the mass error is assessed by comparing the detected mass to charge ratio to the theoretical mass to charge ratio of targeted metabolites. Here, the targeted metabolites have a mass to charge ratio ranging from 74 to 744.

The Y axis here represents the accumulative percentage of metabolites within a certain mass error range. The blue curve shows the result from zero to 12 hours, while the red colored curve shows the data collected from 12 to 24 hours after calibration. More than 90%of the metabolites are within five parts per million mass error, which means the low mass range calibration method developed here is sufficient to maintain five parts per million mass error for low mass range detection.

Another issue to be addressed is the sensitivity of the instrument with the current method and instrument setup. A serial dilution of triplicate samples from a 10 centimeter Petri dish was performed five times with a dilution factor of six resulting in six different concentrations of samples. A targeted list is used to assess the number of metabolites detected at differing concentrations of sample.

The results shown here indicate that the optimal number of targeted metabolites detected is between 2.78 times 10 to the fifth and 1.67 times 10 to the six cells, while one times 10 to the seven cells give a fewer number of detected metabolites, which is due to ion suppression effects. This result indicates that the optimal amount of cells to extract for this analysis is roughly that of a well in a six well plate. For untargeted metabolite analysis, a CV cutoff of 20%and an average intensity value of 10 to the seventh are used to filter the components table.

CV cutoff values can be increased while the average intensity values need to be decreased to include more peaks After manually checking the peaks components with good shapes are selected and searched for in the human metabolome database. The results from data collected in positive mode are listed here, while the results from the negative mode are listed here. Some of the metabolites identified here overlap with the metabolites in the targeted list, such as glutathione and proline.

Additional metabolites absent from the targeted list are also explored, such as methyl glyoxal, which can be derived from glycolysis and one pto, two ELE SN glycerol, three phosphocholine, which is detected in the positive mode with a retention time of 3.2 minutes. After watching this video, you should have a good understanding of how to extract poly metabolites from cultured sars and then use RC QES to measure relative metabolized levels in different samples. Thank you for watching.