calibration of the mass spectrometer for screening lipids with stable isotope labeling by lc-tims-tof ms/ms

Screening Lipid Structures Using Stable Isotope Labeling

When analyzing lipid data, stable isotope labeling (SIL) is an effective method to assess metabolic pathways in living organisms. In this method, atoms within an analyte are replaced by stable isotopes containing 13C or 2H. These isotopes are further incorporated into precursors, which are later embedded within the fatty acids, labeling the areas in which they reside. With these isotopes, one is able to identify the distribution and metabolism of lipids, as they will contrast against the unlabeled background1. Common mass spectrometry platforms are not able to distinguish the signal coming from endogenous molecules2. The success of SIL requires the use of ultrahigh-resolution analytical tools. Liquid chromatography coupled to high-resolution trapped ion mobility spectrometry-parallel accumulation sequential fragmentation-time-of-flight tandem mass spectrometry (LC-TIMS-PASEF-TOF-MS/MS) allows the separation of labeled and nonlabelled species2. The advantage of LC-TIMS-PASEF-TOF-MS/MS analysis is that the MS signal can be filtered by the retention time (RT) and mobility, thus reducing potential interferences of endogenous molecules, as well as quantification and separation of all desired species2. In TIMS, an ion is first held stationary against a gas-mobile phase and subsequently released according to its mobility. This provides high-resolution mobilograms while operating at a lower voltage than earlier IMS instrumentation3. Mobilograms are plots of mass to charge versus drift time that can be used to distinguish between compounds based off of their drift time and amount of overlap4.
By using an additional PASEF technique, sequencing speed is greatly increased without sacrificing sensitivity5. The PASEF theory involves the accumulation of ions followed by their sequential release in the order of their mobility. This is done by accumulating ions in parallel alignment to their mobility. The accumulation in this fashion prevents ion loss. Furthermore, the precursor ion selection occurs simultaneously with the accumulation and release of the ions. With this method, PASEF selects multiple precursors in series during each TIMS scan, rather than the individual precursors per scan typical of a TIMS instrument that does not use PASEF. When the precursor ions are concurrently accumulated and released in 2 ms peaks within a TIMS scan of approximately 100 ms per frame, the signal is amplified by over one order of magnitude, therefore increasing the signal-to-noise ratio3. The PASEF addition to the TIMS increases the efficiency of the MS/MS. As the TIMS-PASEF is coupled with MS/MS, a more efficient fragmentation is possible. Unlike conventional MS/MS detection, the precursor signal is compressed from the TIMS-PASEF, and the fragment ions are detected simultaneously to the TIMS elution time and ion mobility position of the precursor3.
When acquiring data from a mass spectrometer, there are options in the scan mode desired. Data dependent acquisition (DDA) selects precursor ions from the MS1 scan based on their abundances, before fragmenting them and performing the MS2 scan. This scan mode fragments as many precursors as possible. The DDA approach is targeted, and results will depend on the ion selection3. However, DDA-PASEF often has problems in the reproducibility between replicates. While DDA is a great tool in a focused analysis, complex mixtures have greater difficulty in their data acquisition6. This is because only a small amount of ions can be monitored simultaneously3. Data independent acquisition (DIA) is a method where preselected windows of m/z are fragmented independently from their intensity, and all are scanned within the range7. With this scan mode, entire ion clouds are transmitted from the IMS to the mass spectrometer3. In proteomics studies this results in greater amounts of proteins being quantified in a shorter time span compared to DDA. In addition, DIA misses fewer m/z values compared to DDA. Therefore, this alternative has shown great improvements in data reproducibility in signal to noise ratio and better quantification than DDA. In this present work, our goal is to implement DIA to the method reported by Tose et al.2. We improved the reproducibility and intensity of signals, yielding further and more conclusive information on SIL lipids mobilization, distribution and metabolism in biological samples taking advantage of DDA and DIA acquisition.

Author Spotlight: Quantification of Complex Lipidomic Samples Using Stable Isotope Labeling

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

This research intends to implement more specific quantification in lipidomic samples using stable isotope labeling. This allows us to understand lipid panels and highly diverse samples from insects to humans. There currently is a gap in this field regarding the standardization of LC, TIMS, Top, MS/MS in lipidomic 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 team staff, MS/MS, involve DDA and DIA methods.

Calibration of the Mass Spectrometer for Screening Lipids with Stable Isotope Labeling by LC-TIMS-TOF MS/MS

To begin the mass spectrometer, or MS calibration for LC-TIMS-TOF-MS/MS, open the TIMS control software, and under the calibration sub-tab, click on m/z, select tuning mix from the Dropbox, 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 the 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 target's 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 that 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.

Research

JoVE Journal

Chemistry

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable ...