Novel Structure-Driven Predict-to-Hit Strategy for PC Double Bond Positional Isomer Identification Based on Negative LC-MRM Analysis

As the predominant phospholipids in mammalian cells, phosphatidylcholines (PCs) have been demonstrated to play a crucial role in a multitude of vital biological processes. Research has highlighted the significance of the diversity in PC isomers as instigators of both physiological and pathological responses, particularly those with variations in the position of double bonds within their fatty chains. Profiling these PC isomers is paramount to advancing our understanding of their biological functions. Despite the availability of analytical methods utilizing high-resolution secondary mass spectrometry (MS2) fragmentation, a novel approach was imperative to facilitate large-scale profiling of PC isomers while ensuring accessibility, facility, and reliability. In this study, an innovative strategy centered around structure-driven predict-to-hit profiling of the double bond positional isomers for PCs was meticulously developed, employing negative reversed-phase liquid chromatography–multiple reaction monitoring (RPLC-MRM). This novel methodology heightened the sensitivity. The analysis of rat lung tissue samples resulted in the identification of 130 distinct PC isomers. This approach transcended the confines of available PC isomer standards, thereby broadening the horizons of PC-related biofunction investigations. By optimizing the quantitation reliability, the scale of sample analysis was judiciously managed. This work pioneers a novel paradigm for the exploration of PC isomers, distinct from the conventional methods reliant on high-resolution mass spectrometry (HRMS). It equips researchers with potent tools to further explore the biofunctional aspects of lipids.