Comprehensive and high-coverage glycerophospholipidomic analysis based on iterative quadrupole time-of-flight mass spectrometry and its application in cerebral ischemia-reperfusion injury.

Glycerophospholipids play important roles in iron-induced lipid peroxidation during cerebral ischemia-reperfusion, making it essential to investigate changes in their varieties and concentrations under these conditions. However, the wide range of glycerophospholipid contents, particularly the low-abundance species in actual biological samples, posed a challenge for comprehensive analysis. In this study, an iterative quadrupole time-of-flight mass spectrometry (Q-ToF-MS/MS) method was established with the aim of comprehensively detecting glycerophospholipids. This method was a data acquisition strategy implemented through iterative analyses. In each iteration, ions detected in previous runs were excluded, allowing low-abundance glycerophospholipids that were missed by the usual analysis to be extensively detected by a simplified operational process. Using this strategy, 254 glycerophospholipids including 157 PCs, 67 PEs, 19 PGs, 9 PIs, 7 PSs and 5 PAs in rat brain samples were identified after four iterations, and the number of glycerophospholipid species increased by 93.9% compared to a single assay, significantly enhancing the coverage of glycerophospholipid detection. Furthermore, the characteristic fragmentation patterns of six glycerophospholipid subclasses were systematically summarized to improve the accuracy of qualitative identification. In addition, these patterns were also used to construct an ion pair database containing 254 glycerophospholipids, enabling targeted multiple reaction monitoring (MRM) analysis under the optimized high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS) conditions. By comparing the changed glycerophospholipids of rat brains from the normal and cerebral ischemia-reperfusion injury groups, 29 glycerophospholipids were recognized as the potential biomarkers for cerebral ischemia-reperfusion injury, among which nine glycerophospholipids were particularly detected by four iterations. Overall, this iterative MS/MS approach extensively expanded the coverage of low-abundance components, and has been proven to be an effective approach in biomarker screening of cerebral ischemia-reperfusion injury.