"Redox lipidomics technology: Looking for a needle in a haystack"

Abstract

Aerobic life is based on numerous metabolic oxidation reactions as well as biosynthesis of oxygenated signaling compounds. Among the latter are the myriads of oxygenated lipids including a well-studied group of polyunsaturated fatty acids (PUFA) - octadecanoids, eicosanoids, and docosanoids. During the last two decades, remarkable progress in liquid-chromatography-mass spectrometry has led to significant progress in the characterization of oxygenated PUFA-containing phospholipids, thus designating the emergence of a new field of lipidomics, redox lipidomics. Although non-enzymatic free radical reactions of lipid peroxidation have been mostly associated with the aberrant metabolism typical of acute injury or chronic degenerative processes, newly accumulated evidence suggests that enzymatically catalyzed (phospho)lipid oxygenation reactions are essential mechanisms of many physiological pathways. In this review, we discuss a variety of contemporary protocols applicable for identification and quantitative characterization of different classes of peroxidized (phospho)lipids. We describe applications of different types of LCMS for analysis of peroxidized (phospho)lipids, particularly cardiolipins and phosphatidylethanolalmines, in two important types of programmed cell death - apoptosis and ferroptosis. We discuss the role of peroxidized phosphatidylserines in phagocytotic signaling. We exemplify the participation of peroxidized neutral lipids, particularly tri-acylglycerides, in immuno-suppressive signaling in cancer. We also consider new approaches to exploring the spatial distribution of phospholipids in the context of their oxidizability by MS imaging, including the latest achievements in high resolution imaging techniques. We present innovative approaches to the interpretation of LC-MS data, including audio-representation analysis. Overall, we emphasize the role of redox lipidomics as a communication language, unprecedented in diversity and richness, through the analysis of peroxidized (phospho)lipids.

Keywords: Lipid mediators; Mass spectrometry; Oxidatively truncated lipids; Peroxidized phospholipids; Redox lipidomics.

Figure 1.

Structural formulae of major glycerophospholipids and their oxidation products. Glycerophospholipids have two fatty acid residues that are attached at the sn-1, sn-2 positions of the glycerol backbone and polar groups that occupy the sn-3 position. PUFA are usually present at the sn-2 position. OxPLs include a group of oxygenated PUFA-lipids where oxygen-containing functional groups such as hydroperoxy-, hydroxy-, epoxy- and keto- are positioned on the fatty acid in the sn-2 position.

Figure 2.

Schema showing truncation of oxPE and its reaction with nucleophilic amino acids. (a) structure of 1-octadecanoyl-2-(15-hydroperoxy-5E,8E,11E,13E-eicosatetraenoyl)-sn-glycero-3- phosphoethanolamine and possible leaving groups [b; (i)-malondialdehyde, (ii) acrolein, (iii) 4-hydroxyhexaenal, (iv) 4-hydroxynonenal, (v) 4-oxononenal] and remaining groups [c; (i) 1-octadecanoy-2-(15-oxo-5Z,8Z,13E-pentadecatrienoyl)-sn-glycero-3-phosphoethanolamine, (ii) 1-octadecanoy-2-(15, 12-di-oxo-5Z,8Z,13E-pentadecatrienoyl)-sn-glycero-3-phosphoethanolamine, (iii) 1-octadecanoy-2-(15-oxo-12-hydroxy-5Z,8Z,13E-pentadecatrienoyl)-sn-glycero-3-phospho-ethanolamine] electrophiles. (d) reaction schema showing the reaction of oxidized-PE derived electrophiles with neucleophilic amino acid side chains (i) histidine, (ii) cysteine and (iii) lysine

Figure 3.

Structural characterization of oxCL molecular species obtained from ileum of mice exposed to total body irradiation (9.5Gy) using LC-MS/MS Fusion Lumos spectrometer (Thermo Fisher, San Jose, CA). A. Typical base peak profile of CL molecular species with m/z 1465.9755 in the range of retention time (RT) from 15 to 30 min. B. Full MS² spectra of CL molecular species with m/z 1465.9755 at RT 21.8 min (a) and 23.6 min (b). Inserts; MS² spectra in the m/z range from 690 to 720 C. Structural formulas and MS² fragmentation pattern of 13-HODE-CL (a) and 9-HODE-CL (b). D. Full MS³ spectra of CL molecular species with m/z 1465.9755 →295 at RT 21.8 min (a) and 23.6 min (b). Structural formulae and fragmentation patterns of 13-HODE (insert – a) and 9-HODE (insert – b). Mice were exposed to total body irradiation at dose of 9.5Gy. Two days after exposure mice were sacrificed and lipids were extracted by SPE. CLs molecular species were separated by reverse phase chromatography (C18 column was used) and analyzed by ESI-MS/MS using Fusion Lumos (ThermoFisher, San Jose, CA). Two distinct peaks at retention time 21.8 and 23.6 min with m/z 1465.9755 were identified as 13-HODE-CL (left panels) and 9-HODE-CL (right panels)

Figure 4.

A schema illustrating hydrolysis and re-esterification of phospholipids and their oxidation products by PLA₂. Several phospholipases A₂ such as LpPLA₂, calcium independent iPLA₂γ and iPLA₂β release oxidatively modified fatty acids. This approach can significantly reduce the diversification and simplify the initial analysis of oxPL species. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PG, phosphatidylglycerol; PA, phosphatidic acid; CL, cardiolipin; LPC, lyso-phosphatidylcholine; LPE, lyso-phosphatidylethanolamine; LPI, lyso-phosphatidylinositol; LPS, lyso-phosphatidylserine; LPG, lyso-phosphatidylglycerol; LPA, lyso-phosphatidic acid; mCL, mono-lyso-cardiolipin.

Figure 5.

A schema illustrating oxidation and externalization of PS on the surface of apoptotic cells and their engulfment by macrophages. In normal cells, non-oxidized PS is localized exclusively in the inner leaflet of plasma membrane. APT, aminophospholipid translocase.

Figure 6.

Detection and identification of triglycerides and their oxidatively truncated species. A. LC-MS profiles and LC-MS/MS spectra of TAG individual molecular species C16:0/C18:2/C18:1 (a) and their oxidatively truncated derivatives C16:0/C9-ONA/C18:1(b). B. Typical LC-MS profiles and LC-MS/MS spectrum of truncated TAG (C16:0/C9-ONA/C18:1) species before and after incubation with 2,4-dinitrophenylhydrazine, DNPH (a). After derivatization with DNPH the peak at m/z 766 almost disappears (see lower left panel, a) and a new product at m/z 946 is recorded (right upper panel, a). Before TAG derivatization (at the same retention time) the peak at m/z 946 was not observed (right lower panel, a). Structural LC-MS/MS analysis of truncated TAG (C16:0/C9-ONA/C18:1) is shown in panels b and c. Possible structure of the DNPH modified product is inserted. LC-ESI-MS/MS analysis of TAGs/CEs was performed on a Dionex LC system (UltiMate 3000 auto sampler) that was coupled to a Q-Exactive hybrid-quadrupole-orbitrap mass spectrometer (ThermoFisher, Inc. San Jose, CA). TAGs/CEs were separated on a reverse phase column (Luna 3 μm C18 (2) 100A, 150 × 1.0 mm, (Phenomenex)) at a flow rate of 0.065 mL/min. The analysis was performed using gradient solvents (methanol and propanol) containing 0.1% NH₄OH.

Figure 7.

MALDI detection and analysis of molecular species of CL in freshly frozen tissue sections from brains of control rats and after traumatic brain injury (TBI). A. Optical image of an H&E stained coronal rat brain section 3 h after TBI. Point of impact is marked with an arrow. B. MALDI imaging of a serial section from the same brain showing the loss of CL(74:7) from the contusional and peri-contusional areas.