Establishment of a pseudotargeted LC-MS/MS workflow for analyzing triglycerides in biological samples

Background: Triglycerides (TGs) play a crucial role in various physiological processes through the breakdown of their fatty acyl (FA) side chains. It has been demonstrated that not only the total levels of TGs but also the specific composition of FA side chains are vital for biological functions. However, biomedical studies that comprehensively identify FA compositions remain very limited. One of the reasons is the structural heterogeneity of TGs, with variability in their three fatty acyl chains posing significant challenges for TG analysis. Results: This study proposed a pseudotargeted TG analytical workflow that generated a unique dynamic multiple reaction monitoring (dMRM) acquisition list tailored to different biological sample types.TG profiles were acquired in full scan mode using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-qToF), while LC-triple quadrupole mass spectrometry (LC-QqQ) with PIS was applied to identify fatty acyl chains. Finally, dMRM transitions were derived from confirmed ion pairs of TGs with specific FAs. Two demonstration samples, murine type 2 alveolar epithelial cell line, MLE12, with fatty acid synthase deletion, and hypertriglyceridemia plasma, were used to display the capability of the platform. While more TG species were identified in the MLE12 cell samples compared to human plasma samples (53 vs. 47), a more complex and diverse range of FA compositions in TGs was observed in human plasma compared to MLE12 cell samples (379 vs. 167). Significance: Our results emphasize the need for customized MRM acquisition tailored to different biological samples, and the pseudotargeted TG analytical workflow proves effective in improving the understanding of TG regulation in biological systems. This study offers a novel and effective solution to address the complex challenges of TG analysis, enhancing accuracy, specificity, and interpretative strength.