Relative Quantification of Phosphatidylcholine sn-Isomers Using Positive Doubly Charged Lipid–Metal Ion Complexes

Abstract

Phosphatidylcholines are the major phospholipid component of most eukaryotic cell membranes. Phosphatidylcholines have been shown to actively participate in regulatory and metabolic processes. Dysfunctional metabolic processes have been linked to human disease and can result in altered phosphatidylcholine structural features, such as permutation of fatty acid connectivity. Assignment and relative quantitation of structural isomers that arise from fatty acid permutation on the phosphatidylcholine backbone, so-called sn-isomers, is difficult with routine tandem mass spectrometry or with liquid chromatography without authentic standards. In this work, we report on the observation that phosphatidylcholines form abundant doubly charged metal ion complexes during electrospray ionization (ESI) and show that these complexes can be used to assign fatty acid moieties, relatively quantify sn-isomers in MS experiments, and mass spectrometrically separate phosphatidylcholines from other phospholipid classes in positive ion mode. Addition of Fe salts (20 mol %) to ESI spray solutions affords highly abundant doubly charged metal ion phosphatidylcholine complexes (∼110% of protonated compounds) and allows sensitive fragment ion detection (limit of detection = 100 pM). Higher energy collisional dissociation, collision-induced dissociation, and ultraviolet photodissociation of doubly charged complexes yield two fragment ions for every fatty acid moiety. The latter two tandem MS methods preferentially yield sn-2 associated product ions enabling relative sn-isomer quantification. The analytical utility of doubly charged phosphatidylcholine-metal ion complexes is demonstrated for polar lipid extracts, including extracts from diabetes type 1 and type 2 mouse models, and sn-isomer abundances are derived.

Unveiling Glycerolipid Fragmentation by Cryogenic Infrared Spectroscopy

“…Different stabilities of fatty acyls depending on their sn -position have been observed in other studies and are being exploited for the distinction of sn -isomers in glycerophospholipids. 16 , 17 The crucial role of alpha hydrogens in glycerolipid fragmentation was later confirmed for triacylglycerols lacking hydrogens at the α position. 18 …”

Large-scale lipid analysis with C=C location and sn-position isomer resolving power

“…PB reaction converts the C=C to an oxetane which can be preferentially fragmented by low-energy collisioninduced dissociation (CID). Other approaches include ion mobility spectrometry (IMS) 36,37 and alternative gas-phase ion activation [38][39][40][41][42][43][44] . These lipid characterizing methods were also compatible with mass spectrometry imaging (MSI) [45][46][47][48][49][50] .…”

Advanced tandem mass spectrometry in metabolomics and lipidomics—methods and applications

“…In addition to product ions from the saccharide moieties of these compounds, diagnostic ions for FA side chains and the sphingoid base helped to extensively characterize the structure of these complex lipids. Follow-up studies showed that optimized experimental settings enable identification of DB positions [77,78], sn-isomers [77,79,80], hydroxylation sites as well as linkages [81], and FA branching/ cylclopropanation [82] sites in glycerophospholipids and sphingolipids when using 193 nm or 213 nm UVPD. All these studies showed that UVPD results in extensive metabolite and lipid fragmentation, thereby enabling annotation of structural details not accessible with CID methodologies.…”