Journal
JOURNAL OF LIPID RESEARCH
Volume 61, Issue 12, Pages 1707-1719Publisher
ELSEVIER
DOI: 10.1194/jlr.RA120001044
Keywords
collision-induced dissociation; acyl chain; ether phospholipids; cardiolipin; odd-numbered acyl chain; high-performance liquid chromatography-tandem mass spectrometry; lipidomics
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Funding
- National Institutes of Health [PO1 AG015885, R21 AG049461]
- Center for Mitochondrial Diseases, Case Western Reserve University
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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.
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