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

Phospholipids are major components of most eukaryotic cell membranes. Changes in metabolic states are often accompanied by phospholipid structure variations. The structural changes of phospholipids are the hallmark of disease states, or specific lipid structures have been associated with distinct organisms. Prime examples are microorganisms that synthesize phospholipids with, for example, different branched chain fatty acids. Assignment and relative quantitation of structural isomers of phospholipids that arise from attachment of different fatty acids to the glycerophospholipid backbone are difficult with routine tandem mass spectrometry or with liquid chromatography without authentic standards. In this work, we report on the observation that all investigated phospholipid classes form doubly charged lipid-metal ion complexes during electrospray ionization (ESI) and show that these complexes can be used to assign lipid classes and fatty acid moieties, distinguish isomers of branched chain fatty acids, and relatively quantify these isomers in positive-ion mode. Use of water free methanol and addition of divalent metal salts (100 mol %) to ESI spray solutions afford highly abundant doubly charged lipid-metal ion complexes (up to 70 times of protonated compounds). Higher-energy collisional dissociation and collision-induced dissociation of doubly charged complexes yield a diverse set of lipid class-dependent fragment ions. In common for all lipid classes is the liberation of fatty acid-metal adducts that yield fragment ions from the fatty acid hydrocarbon chain upon activation. This ability is used to pinpoint sites of branching in saturated fatty acids and is showcased for free fatty acids as well as glycerophospholipids. The analytical utility of doubly charged phospholipid-metal ion complexes is demonstrated by distinguishing fatty acid branching-site isomers in phospholipid mixtures and relatively quantifying the corresponding isomeric compounds.