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.

总甘油三酯 (TAG) 水平是代谢和心血管疾病的关键临床标志物。然而，由于其极端的结构复杂性，个别 TAG 的作用尚未得到彻底探索。我们提出了一种结合多反应监测 (MRM) 和多级质谱 (MS ³ ) 的靶向质谱方法，用于对 TAG 进行全面的定性和半定量分析。这种方法称为 TriP-MS3 - 使用 MS ³进行三酰基甘油分析- 以全自动方式筛选 6,700 多种 TAG 物种。TriP-MS3 表现出出色的重现性（中位日间 CV ∼ 0.15）和线性（中位 R ² = 0.978) 并在人血浆中检测到 285 种单独的 TAG。通过与最先进的反相液相色谱 (RPLC)-MS (R ²  = 0.83) 进行比较，验证了该方法的半定量准确性，这是最常用的 TAG 分析方法。最后，我们通过表征脂肪酸去饱和酶抑制剂在体外对 TAG 谱的影响以及通过分析秀丽隐杆线虫中的 TAG 来证明该方法的实用性和多功能性。