Abstract
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 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 PS, containing an ether linkage and species containing odd-numbered acyl chains in cardiolipin, PS, PI, and PG. 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.
Highlights
IntroductionDifferent combinations of the head groups and the acyl chains give rise to numerous isobaric and isomeric species of phospholipids
Supplementary key words collision-induced dissociation acyl chain ether phospholipids cardiolipin odd-numbered acyl chain highperformance liquid chromatography-tandem mass spectrometry lipidomics
There is a chance for misidentification between isobaric species, especially when unusual or unexpected species such as ether phospholipids or phospholipids with odd-numbered acyl chains are present
Summary
Different combinations of the head groups and the acyl chains give rise to numerous isobaric and isomeric species of phospholipids Using this concept and commonly observed acyl chains, assignments of peaks based on their masses are generally accepted for global lipidomics [2,3,4]. This type of analysis does not differentiate between isomeric species and neglects the importance of individual species. Our data markedly differ in the assignments of phospholipid classes and species compared with those recently made in MALDI-TOF lipidomics of BHMs [2] They labeled six species of PS, which were not contained in our PS class, and we identified these as ether-linked PE.
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