Formation and Tandem Mass Spectrometry of Doubly Charged Lipid-Metal Ion Complexes

Phospholipids are major components of most eukaryoticcell membranes.Changes in metabolic states are often accompanied by phospholipidstructure variations. The structural changes of phospholipids arethe hallmark of disease states, or specific lipid structures havebeen associated with distinct organisms. Prime examples are microorganismsthat synthesize phospholipids with, for example, different branchedchain fatty acids. Assignment and relative quantitation of structuralisomers of phospholipids that arise from attachment of different fattyacids to the glycerophospholipid backbone are difficult with routinetandem mass spectrometry or with liquid chromatography without authenticstandards. In this work, we report on the observation that all investigatedphospholipid classes form doubly charged lipid-metal ion complexesduring electrospray ionization (ESI) and show that these complexescan be used to assign lipid classes and fatty acid moieties, distinguishisomers of branched chain fatty acids, and relatively quantify theseisomers in positive-ion mode. Use of water free methanol and additionof divalent metal salts (100 mol %) to ESI spray solutions affordhighly abundant doubly charged lipid-metal ion complexes (up to 70times of protonated compounds). Higher-energy collisional dissociationand collision-induced dissociation of doubly charged complexes yielda diverse set of lipid class-dependent fragment ions. In common forall lipid classes is the liberation of fatty acid-metal adducts thatyield fragment ions from the fatty acid hydrocarbon chain upon activation.This ability is used to pinpoint sites of branching in saturated fattyacids and is showcased for free fatty acids as well as glycerophospholipids.The analytical utility of doubly charged phospholipid-metal ion complexesis demonstrated by distinguishing fatty acid branching-site isomersin phospholipid mixtures and relatively quantifying the correspondingisomeric compounds.

Automated summary

Phospholipids are crucial components of eukaryotic cell membranes and undergo structural changes during metabolic states. These structural variations are associated with disease states and specific organisms. This study demonstrates that all analyzed phospholipid classes form doubly charged lipid-metal ion complexes during electrospray ionization, which can be utilized for lipid class assignment and fatty acid identification. The use of water free methanol and divalent metal salts greatly enhances the abundance of these complexes. This method allows for the differentiation of fatty acid isomers and relative quantitation of these isomeric compounds.