Phospholipids, including ether phospholipids, are composed of numerous isomeric and isobaric species that have the same backbone and acyl chains. This structural resemblance results in similar fragmentation patterns by collision-induced dissociation of phospholipids regardless of class, yielding complicated tandem mass spectrometry (MS/MS) spectra when isobaric species are analyzed together. Furthermore, the presence of isobaric species can lead to misassignment of species when made solely based on their molecular weights. In this study, we used normal-phase HPLC for ESI-MS/MS analysis of phospholipids from bovine heart mitochondria. Class separation by HPLC eliminates chances for misidentification of isobaric species from different classes of phospholipids. Chromatography yields simple MS/MS spectra without interference from isobaric species, allowing clear identification of peaks corresponding to fragmented ions containing monoacylglycerol backbone derived from losing one acyl chain. Using these fragmented ions, we characterized individual and isomeric species in each class of mitochondrial phospholipids, including unusual species such as phosphatidylserine containing an ether linkage and species containing odd-numbered acyl chains in cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. We also characterized monolysocardiolipin and dilysocardiolipin, the least abundant, but nevertheless important mitochondrial phospholipids. The results clearly show the power of HPLC-MS/MS for identification and characterization of phospholipids including minor species.

中文翻译：

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通过 HPLC-MS/MS 鉴定牛心脏线粒体中的异常磷脂。


磷脂，包括醚磷脂，由许多具有相同主链和酰基链的异构体和同量异位物质组成。这种结构相似性会导致无论何种类别的磷脂都会因碰撞引起的解离而产生相似的碎片模式，当同时分析同量异位物质时，会产生复杂的串联质谱 (MS/MS) 谱图。此外，当仅根据其分子量进行分类时，同量异位物质的存在可能会导致物质的错误分配。在本研究中，我们使用正相 HPLC 对牛心脏线粒体磷脂进行 ESI-MS/MS 分析。通过 HPLC 进行类别分离，消除了错误识别不同类别磷脂中同量异位物质的可能性。色谱法产生简单的 MS/MS 谱图，不受同量异位物质的干扰，从而可以清楚地识别与含有因失去一条酰基链而产生的单酰基甘油主链的碎片离子相对应的峰。使用这些碎片离子，我们对每一类线粒体磷脂中的个体和异构体进行了表征，包括不常见的种类，例如含有醚键的磷脂酰丝氨酸，以及心磷脂、磷脂酰丝氨酸、磷脂酰肌醇和磷脂酰甘油中含有奇数酰基链的种类。我们还鉴定了单溶心磷脂和二溶心磷脂，它们是含量最少但仍然重要的线粒体磷脂。结果清楚地表明了 HPLC-MS/MS 在鉴定和表征磷脂（包括少数种类）方面的能力。