Phosphatidylglycerol (PG) and phosphatidylinositol (PI) are two essential classes of glycerophospholipids (GPs), playing versatile roles such as signalling messengers and lipid-protein interaction ligands in cell. Although a majority of PG and PI molecular species contain unsaturated fatty acyl chain(s), conventional tandem mass spectrometry (MS/MS) methods cannot discern isomers different in carbon-carbon double bond (CC) locations. In this work, we paired phosphate methylation with acetone Paternò-Büchi (PB) reaction, aiming to provide a solution for sensitive and structurally informative analysis of these two important classes of GPs down to the location of CC. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow was established. Offline methylated PG or PI mixtures were subjected to hydrophilic interaction chromatographic separation, online acetone PB reaction, and MS/MS via collision-induced dissociation (CID) for CC location determination in positive ion mode. This method was sensitive, offering limit of identification at 5 nM for both PG and PI standards down to CC locations. On molecular species level, 49 PI and 31 PG were identified from bovine liver, while 61 PIs were identified from human plasma. This workflow also enabled ratiometric comparisons of CC location isomers (C18:1 Δ9 vs. Δ11) of a series of PIs from type 2 diabetes (T2D) plasma to that of normal plasma samples. PI 16:0_18:1 and PI 18:0_18:1 were found to exhibit significant changes in CC isomeric ratios between T2D and normal plasma samples. The above results demonstrate that the developed LC-PB-MS/MS workflow is applicable to different classes of lipids and compatible with other established lipid derivatization methods to achieve comprehensive lipid analysis.

中文翻译：

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C=C 位置水平上磷脂酰肌醇和磷脂酰甘油的脂质组表征

磷脂酰甘油 (PG) 和磷脂酰肌醇 (PI) 是甘油磷脂 (GP) 的两个基本类别，在细胞中发挥着多种作用，例如信号信使和脂蛋白相互作用配体。尽管大多数 PG 和 PI 分子种类包含不饱和脂肪酰基链，但传统的串联质谱 (MS/MS) 方法无法辨别碳碳双键 (CC) 位置不同的异构体。在这项工作中，我们将磷酸甲基化与丙酮 Paternò-Büchi (PB) 反应配对，旨在为这两种重要类别的 GP 直至 CC 位置的敏感和结构信息分析提供解决方案。建立了液相色谱-串联质谱 (LC-MS/MS) 工作流程。离线甲基化 PG 或 PI 混合物经过亲水相互作用色谱分离、在线丙酮 PB 反应和 MS/MS 通过碰撞诱导解离 (CID) 在正离子模式下进行 CC 定位。这种方法很灵敏，对于 PG 和 PI 标准，低至 CC 位置的识别限为 5 nM。在分子种水平上，从牛肝中鉴定出 49 种 PI 和 31 种 PG，而从人血浆中鉴定出 61 种 PI。该工作流程还支持对来自 2 型糖尿病 (T2D) 血浆和正常血浆样品的一系列 PI 的 CC 位置异构体（C18:1 Δ9 与 Δ11）进行比例比较。发现 PI 16:0_18:1 和 PI 18:0_18:1 在 T2D 和正常血浆样品之间的 CC 异构体​​比率上表现出显着变化。