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. 2021 Nov 1:1184:339023.
doi: 10.1016/j.aca.2021.339023. Epub 2021 Sep 3.

In-depth triacylglycerol profiling using MS3 Q-Trap mass spectrometry

Matias Cabruja  1 , Josefina Priotti  2 , Pablo Domizi  3 , Katharina Papsdorf  1 , Deanna L Kroetz  2 , Anne Brunet  1 , Kévin Contrepois  4 , Michael P Snyder  5
Affiliations

In-depth triacylglycerol profiling using MS3 Q-Trap mass spectrometry

Matias Cabruja et al. Anal Chim Acta. .

Abstract

Total triacylglycerol (TAG) level is a key clinical marker of metabolic and cardiovascular diseases. However, the roles of individual TAGs have not been thoroughly explored in part due to their extreme structural complexity. We present a targeted mass spectrometry-based method combining multiple reaction monitoring (MRM) and multiple stage mass spectrometry (MS3) for the comprehensive qualitative and semiquantitative profiling of TAGs. This method referred as TriP-MS3 - triacylglycerol profiling using MS3 - screens for more than 6,700 TAG species in a fully automated fashion. TriP-MS3 demonstrated excellent reproducibility (median interday CV ∼ 0.15) and linearity (median R2 = 0.978) and detected 285 individual TAG species in human plasma. The semiquantitative accuracy of the method was validated by comparison with a state-of-the-art reverse phase liquid chromatography (RPLC)-MS (R2 = 0.83), which is the most commonly used approach for TAGs profiling. Finally, we demonstrate the utility and the versatility of the method by characterizing the effects of a fatty acid desaturase inhibitor on TAG profiles in vitro and by profiling TAGs in Caenorhabditis elegans.

Keywords: Fatty acid; Lipids; Mass spectrometry; Triacylglycerols.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1.
Figure 1.. Data acquisition principies of TriP-MS3.
(a) Simplified schematic representation of species produced from MRM and MS3 fragmentation experiments. MRM produces [M-(FA+NH3)]+ following the neutral loss of one FA and MS3 fragmentation of this ion generates up to 4 ions called [RCO]+ and [MAG-H2O]+ for each remaining 2 FAs. (b) TriP-MS3 identification and semiquantification workflow using TAG 50:1 as an example. Following the neutral loss of FA 16:0, the MS3 spectrum present signal from 6 individual FAs leading to the identification of 3 individual TAG species TAG 16:0_16:0_18:1, TAG 16:0_16:1_18:0 and TAG 16:0_14:0_20:1. For simplicity, only the most abundant [RCO]+ ions are represented for each FA. The TAG feature TAG 50:1-FA 16:0 concentration is estimated at the MRM level using the spiked-in deuterated TAG standard. TAG species concentrations are estimated using the relative abundance of signals from FAs at the MS3 level.
Figure 2.
Figure 2.. Three-dimensional representation of individual TAG species identified in human plasma by TriP-MS3 (n = 285).
Each dot is colored by its concentration and represents a TAG species composed of the FAs indicated on the axes.
Figure 3.
Figure 3.. Technical performances of TriP-MS3.
(a) Number of TAG species and features identified at different human plasma concentrations. (b) TAG signal linearity reported with the Pearson coefficient of determination (R2) calculated across two concentration ranges. The lower concentration range (0.01–10X) has 18 species and 127 features. The higher concentration range (0.5–10X) has 249 species and 254 features. (c) Dilution curve for TAG 18:1_18:0_16:0 across the whole range of concentrations. Each sample was analyzed in technical triplicates. (d) Intra- and inter-day variability was calculated on technical triplicates the same day and using the mean of each day, respectively. All TAG species (n = 285) and TAG features (n = 289) detected in 1X plasma were used in the analysis.
Figure 4.
Figure 4.. Comparison of TriP-MS3 semiquantitative measurements with other methods.
(a) Abundance of TAG groups in human plasma analyzed by three different approaches: TriP-MS3, LC-MS and MRM. TAG group abundances were calculated by summing the estimated concentrations of all TAG species and TAG features containing the same number of carbons and unsaturations. The bars represent the mean of biological triplicates (same sample prepared three times), the individual values are shown as dots. Lines connecting the mean measurement of each method were added to help compare the different approaches. (b) Correlation of semiquantitative measurements obtained with TriP-MS3 and LC-MS methods using the top 50 TAG species detected in human plasma. Average values from biological triplicates were used.
Figure 5.
Figure 5.. TAG profile changes following treatment with a desaturase inhibitor.
Changes at FA composition (a) and TAG group levels (b) in HepG2 cells treated with a Δ5/Δ6 desaturase inhibitor (CP-24879). The abundance of each TAG FA was calculated by summing the estimated concentration of all TAG features and all TAG species containing a given FA. For TAG species with one FA present more than once, the concentration was multiplied by the number of times the FA was present in the molecule. TAG group abundances were calculated by summing the estimated concentration of all TAG species and TAG features containing the same number of carbons and unsaturations. Species that increased or decreased with CP-24879 treatment were colored in red and blue, respectively. Biological sextuplicates were used in the analysis and only species with significant changes (Two-tailed Mann-Whitney, adjusted (Benjamini–Hochberg) p-value < 0.05) were shown. Fold changes of analytes detected in only one condition (infinite fold change) were plotted as fold change = 3.
Figure 6.
Figure 6.. Comparison of TAG coverage using the MRM and TriP-MS3 methods.
Relative abundance of TAG FAs in human plasma (a) and in C. elegans lipid droplets (b). The abundance of each TAG FA was calculated by summing the estimated concentration of all TAG features and all TAG species containing the FA. For TAG species with one FA present more than once, the concentration was multiplied by the number of times the FA was present in the molecule. The concentrations were then normalized to the sum of all the species detected. The bars represent the mean of biological triplicates (same sample prepared in triplicates) and the individual values are shown as dots.
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