Identification and quantitation of lipid C=C location isomers: A shotgun lipidomics approach enabled by photochemical reaction

Abstract

The field of lipidomics has been significantly advanced by mass spectrometric analysis. The distinction and quantitation of the unsaturated lipid isomers, however, remain a long-standing challenge. In this study, we have developed an analytical tool for both identification and quantitation of lipid C=C location isomers from complex mixtures using online Paternò-Büchi reaction coupled with tandem mass spectrometry (MS/MS). The potential of this method has been demonstrated with an implementation into shotgun lipid analysis of animal tissues. Among 96 of the unsaturated fatty acids and glycerophospholipids identified from rat brain tissue, 50% of them were found as mixtures of C=C location isomers; for the first time, to our knowledge, the quantitative information of lipid C=C isomers from a broad range of classes was obtained. This method also enabled facile cross-tissue examinations, which revealed significant changes in C=C location isomer compositions of a series of fatty acids and glycerophospholipid (GP) species between the normal and cancerous tissues.Paternò-Büchi reaction | glycerophospholipids | photochemical reaction | lipid biomarkers | cancerous tissue analysis L ipids play a multitude of crucial roles in biological systems by serving as building blocks of cell membranes, sources for energy storage, and media for signal transduction (1-3). Unveiling the mechanisms and networks behind lipid homeostasis calls for sensitive, quantitative, and molecularly specific lipid analysis (4). The recent advancement in mass spectrometry (MS) for bioanalysis has enabled the field of lipidomics (5, 6) by allowing global identification and quantitation of lipid species at high speed (7-9) and providing information of lipid-lipid (10, 11) and lipidprotein interactions (12, 13) at systems level. These capabilities further expedite research on lipid biomarker discovery and metabolite flux analysis (14-16). Among many analytical figures of merit, high molecular specificity is a distinct feature of the MSbased approaches. Rich structural information of lipids in complex biological samples can now be routinely obtained, including the classes of the lipids, fatty acyl/alkyl composition, and even the sn positions of the fatty acyl/alkyl chains (17-19). The locations of the carbon-carbon double bonds (C=C) in the lipids, however, have rarely been identified using commercial MS systems and therefore have been either assumed or not reported in a large body of literatures for lipid study (20).The MS/MS methods, especially those involving low-energy collision-induced dissociation (CID), have not been effective in locating C=C bond locations, which is due to the high bond dissociation energies associated with cleaving a C=C bond. Without characteristic fragment ions produced, the C=C locations cannot be determined using MS/MS. To tackle this problem, two MS approaches have been explored, each with successes achieved but also with limitations observed. The first one employs C=C specific chemical derivatizations before MS analysis. T...

Collision-induced dissociation of doubly-charged barium-cationized lipids generated from liquid samples by atmospheric pressure matrix-assisted laser desorption/ionization provides structurally diagnostic product ions

“…In CID product ion spectra of lipids, a characteristic Δm/z of 26 per double bond indicates their position in the acyl chains [29]. Relative and absolute quantitation of constituent isomers has been reported based on the intensity of the diagnostic product ions [30]. Recently, this characteristic Δm/z of 26 has been applied to double bond determination in cholesteryl ester lithium adducts by MS 3 analysis of the PB-reaction products [31].…”

Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics (EIEIO)

“…Generally, after a TAG ion is fragmented using conventional CID, acyl chain fragment losses can be detected, and the degree of total unsaturation within those acyl chains can be estimated (6). However, the identification of the specific locations of these carbon-carbon double bonds is much more difficult, requiring the use of ion/molecule reactions (11)(12)(13)(14). With EIEIO, we are able to perform this identification in TAG molecules much the same as we previously reported for PCs (18) (Figs.…”

“…However, a distorted V-shaped profile still appears at the double bond locations, making double bond identification possible. For example, in the With the preliminary DMS conditions set for the targeted TAG precursors, further refinement of the DMS conditions (step 5) are found for each targeted TAG precursor to obtain the most intense precursor ions with the lowest levels of interference from species such as 13 C isotopomers and oxidized species. The first choice should be lower DR value because it provides better ion transmission with shorter EIEIO accumulation time required.…”

Pinpointing Double Bond and sn-Positions in Glycerophospholipids via Hybrid 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry

“…32–33 Promising new methods to elucidate double bond position(s) include Paternò-Büchi (PB) reactions, 34–36 radical-directed dissociation (RDD) via 266 nm photodissociation followed by CID, 37 metastable atom activated dissociation (MAD), 38 charge transfer dissociation (CTD) 39 and charge reversal and charge-switch derivatization. 40–41 Ozone-induced dissociation (OzID), 42–43 which utilizes ion-molecule reactions between ionized, unsaturated lipids and neutral ozone within a mass spectrometer, is a clever strategy to assign double bond position, but reaction times of ~0.2 – 10 s are less amenable for high throughput chromatographic application.…”