Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS

This research intends to implement more specific quantification in lipidemic samples using stable isotope labeling. This allows us to understand lipid panels in highly diverse samples from insects to humans. There currently is a gap in this field regarding the standardization of LC-TIMS-TOF MS/MS in lipidemic investigations.

Here, we provide a method for tracking biomass remains for this purpose. In the future, our research will involve the investigation of epigenetics and metabolomics present in the biological process using the LC-TIMS-TOF MS/MS involve DDA and DIA methods. To begin the mass spectrometer or MS calibration for LC-TIMS-TOF MS/MS, open the TIMS control software, and under the calibration subtab, click on M over Z, select tuning mix from the drop box, and click calibrate at the bottom right corner.

Continue to click calibrate until a score of 100%is achieved. Now select the mobility tab and follow the same steps as demonstrated until the 100%calibration score is reached. To generate each calibration point in the calibration curve, spike the lipid internal standard mixture with 10 microliters of deuterated internal standard mixture.

Prepare seven such spiked samples for seven calibration points in the desired range. Using the data analysis software, manually annotate the fatty acid chains based on the passive MS/MS patterns. To determine that stable isotope labeling or SIL efficiency, calculate the ratio of the area of the SIL containing isotopes to the area of the non-SIL containing isotopes.

Calculate the theoretical pattern by determining the probability of finding a labeled atom at any site. Calculate SIL enrichment by fitting the experimental isotope profiles with the theoretical pattern simulated on data analysis software. To start the run, activate the column oven by clicking the thermometer button and activate the pumps by clicking the valve button.

Set the MS method to the appropriate method saved earlier. Verify that the injection volume is five microliters and line one targets vial 20 to one and save the sample list. Start the sequence and confirm successful injection by the message stating injected displayed on the system.

Copy line one and paste it below to generate a new line two. Populate line two with samples depending on the experiment, ensure that the correct position in the autosampler is used. If the mobile phases are fresh, first, perform a peak shape test with the lipid mixture standards from the manufacturer to compare it with previous results.

The lipid mixture and the standards are displayed on the screen. Make sure new lines have the correct separation method and not the preconditioning method used in line one. Ensure the MS method entered is correct.

Check that the correct processing method is loaded and the run automated process is enabled. Save the sample list to ensure samples are analyzed after the current run finishes. To begin, open the data analysis software.

At the top left corner, click on the file button and select open. To select the files of choice, right click on the file name and select properties. Then, select calibration status.

At the dropdown box, select internal calibration for the mass spectrometer. Ensure that all errors are less than one parts per million. After that, select internal mobility calibration and confirm that the error is less than one part per million.

Next, right click on chromatogram and select edit chromatogram. At type, click the dropdown box and select extracted ion chromatogram. Under filter, select all MS, and for scan mode, select all to view the peaks for the molecule of interest rather than only its fragments.

There are two filter options for molecule selection, masses or formula. If masses is selected, choose the theoretical M over Z of the molecule of interest, which in this case is 829.7985. If formula is selected, choose the formula for the molecule and the ion forms of interest, which in this case is ammonium.

For width, select plus or minus one. For polarity, ensure that it maintains the positive mode. Under mobility, select 1.455 to 1.465 to isolate the molecule of interest and filter out molecules not falling within these values.

Once the parameters are selected, click change, followed by OK at the top right. After a brief time, the software will process the selections and output a chromatogram. Right click at the baseline of the origin of the peak of interest and drag while holding to the end of the peak to obtain a live mass spectrum of fragments within that retention time.

Right click at the left of the screen on mobilogram and select edit mobilogram. A window named edit mobilogram traces will appear. Follow the steps demonstrated previously.

For retention time, input the retention time of the peak of interest. Once the parameters are selected, click add, and OK at the top right. Wait till the software processes the selections and outputs a chromatogram.

For extracting ions, repeat the steps demonstrated previously until a list of ions is formed. At the bottom of the MS window, select profile MS and fragment MS to allow a spectrum view of ions from a full scan in passive. For the spectrum view, right click and select copy compound spectrum.

The spectrum data that appears at the right can provide more information on the compound, such as resolution, resolving power, intensity, and signal to noise ratio. To process the data, manually integrate the chromatogram and mobility peaks to yield valuable information on the molecule of interest. Right click find and select compounds manually chromatogram or mobilogram.

Left click and highlight the desired peak. The obtained information includes retention time, area, signal to noise ratio, and mobility.