The overall goal of the following experiment is to compare snapshots of the metabolic profiles of different groups of biologic samples. This is achieved by using a liquid, liquid extraction to separate organic and polar molecules. As a second step molecules are further separated by liquid chromatography and analyzed by high resolution mass spectrometry, which provides retention time, mass to charge ratio, and intensity data for further analysis.
Next liquid chromatography runs are aligned and features are detected, quantified, and sorted. In order to identify differentially abundant features between groups. Results show the differences in features across treatment conditions based on differential abundance of analytes as detected by liquid chromatography, high resolution mass spectrometry.
Although this method can be used to study cellular metabolism, it can also provide insight into biomarker discovery or zeno biotic metabolism. For adherent lines, add 1.5 milliliters of media to the cells in a 10 centimeter plate. Place the plate on ice and gently scrape to lift the cells.
Transfer the 1.5 milliliters of lifted cell suspension to a pre-labeled 10 milliliter glass centrifuge tube. Next, add six milliliters of two to one chloroform methanol to each of the 10 milliliter glass tubes containing the samples. Shake the samples on low speed for 30 minutes After shaking, centrifuge the samples with a low acceleration deceleration setting at 1, 935 times G for 10 minutes at four degrees Celsius to completely separate the phases using a long stem pasture pipette.
Transfer the organic layer to a new pre-labeled 10 milliliter glass centrifuge tube. Next, transfer the aqueous layer into a clean plastic two milliliter einor tube. At this point, evaporate the organic and aqueous samples under nitrogen gas.
Reconstitute the dried down samples in 50 to 100 microliters of the starting solution for the desired UPLC method. Gently pipette the sample up and down to aid in dissolving the analytes. Then transfer each sample into a 0.22 micron nylon tube filter and spin up to 14, 000 times G for approximately five minutes until the sample has completely passed through the filter.
Inspect the sample to ensure that there are no visible precipitates remaining in the sample. Transfer the supernatant of the filtered sample into a pre-labeled UPLC vial with insert cap the vial. Then flick it to remove any bubbles from the bottom of the sample vial.
Place the sample in the chilled autos sampler. Now create a run for the organic sample using the control software for the liquid chromatograph. Following this, create a run for the organic sample based off of the optimized UPLC conditions.
Use the optimized UPLC conditions to create a method for the aqueous phase as well. If a known set of target analytes is of high interest, optimize the UPLC conditions using the heavy labeled analog of these compounds and generate a method. Using these conditions, allow the UPLC to adequately equilibrate.
This should include a full priming of solvents Before attaching a column and following the manufacturer's directions for equilibration volume of a new column, maintain a log of starting back pressures to help diagnose future problems using the control software for the high resolution mass spectrometer. Create a positive mode method. Then using the same conditions, create a negative mode method.
Clean and calibrate the instrument. Establish a stable spray into the spectrometer and allow time for the calibration solution to adequately dissipate from the source and optics. Acceptable stability of spray should give a three to 5%relative standard deviation of intensity over at least 100 scans with injection of calibration solution at the same flow rate as the lc method being used.
Using a random number generator and a key set up samples in a randomized manner to avoid any batch effects introduced by the analysis. Set up the sequence for all the samples. Set appropriate injection volume and run a blank before the first sample.
If carryover or matrix effects between samples are suspected, run adequate blanks or washes, begin sequence and monitor periodically for problems including pressure fluctuations or loss of signal intensity across the run. If severe problems are detected, stop the run. Clean and calibrate the instrument.
Establish a stable spray into the spectrometer and allow time for the calibration solution to adequately dissipate from the source and optics. Before beginning differential analysis, manually check the total ion currents or look at filtered chromatograms for any known compound in the sample. To ensure reproducibility of runs and injection slash UPLC slash spray stability throughout all the samples, transfer the RAW data files from the mass spectrometer to the hard drive of the workstation where sieve is installed.
Open sieve. Begin a new experiment and load the appropriate files into sieve. Assign comparison groups and select a single reference file to allow SIE V to generate the parameters for analysis.
Follow the prompts and adjust any parameters per requirements of any individual experiment. Load the correct adduct list for positive or negative mode in the parameters. Once the analysis is finished, check that a list of frames has been populated on the screen.
Ensure that the lc alignment overlays any landmark peaks. If any of the unaligned lc runs show large deviation in landmark peaks. This may indicate a problem with the sample.
Plot the data by coefficient of variation within a like sample population. Sort the list based on desired traits. Next, examine each peak for appropriate peak alignment across groups.
Peak integration, Gaussian, peak shape signal intensity, isotopes and adduct assignment. Export data as desired for further analysis or to generate targeted mass lists for confirmation and MS to the end experiments. A large number of hits were identified by the two programs used for differential green dots are features more abundant in the control, whereas red dots are more abundant in the treatment.
A larger dot size indicates an increased magnitude of fold change between the groups and the intensity of the color demonstrates a decreasing P value with increasing saturation of color pictured. Here are the chromatograms showing the organic phase positive ion mode UPLC MS analysis window A shows the total ion current from S Civ 2.0. The extracted ion chromatogram for the feature identified as D paneth by XCMS is displayed in window B.Window C shows the extracted ion chromatogram for the feature identified as rot known from sve.
Window D shows the extracted ion chromatogram for the feature identified as rot known from sve normalized to the total ion current. A differentially abundant feature reduced in the rot known treated group was tentatively identified as d panadine by accurate mass database searching. The identity of the analyte was confirmed with U-P-L-C-H-R tandem mass spectrometry.
Shown here is a comparison of the chromatograms and mass spectra of the sample and the pure compound. Don't forget that working with chloroform can be extremely hazardous and proper precautions such as a well ventilated fume hood should be followed.
