Deep Lipidomics and Molecular Imaging of Unsaturated Lipid Isomers: A Universal Strategy Initiated by mCPBA Epoxidation

Abstract

Biosynthesis of unsaturated lipids is highly regulated through cellular lipogenesis, rendering diverse double-bond positional isomers (C=C isomers) of a given unsaturated lipid species. However, identification and quantification of C=C isomers are usually challenging using conventional mass spectrometric analyses as they yield indistinguishable tandem mass spectra. To address this challenge, we proposed an easy-to-use, cost-effective, handy derivatization method, MELDI (mCPBA Epoxidation for Lipid Double-bond Identification), and its integrations with liquid chromatography mass spectrometry (LC-MS) lipidomics as well as desorption electrospray ionization (DESI) mass spectrometry imaging (MSI). Using MELDI-LC-MS, we identified more than 100 C=C isomers among mono-and poly-unsaturated fatty acids and glycerolphospholipids in human serum, where a variety of uncommon C=C isomers with low endogenous levels were quantified. The capability of MELDI-LC-MS in lipidome analysis was also demonstrated by the differentiated 3T3-L1 adipocytes, providing an insight into the cellular lipid reprogramming upon stearoylcoenzyme A desaturase 1 (SCD1) inhibition at the C=C isomer level. Additionally, combining MELDI with DESI-MSI allowed us to spatially resolve the tumor-associated change in C=C isomers in a metastatic mouse lung tissue section. Our results suggested that C=C isomers are potential indicators to disease-related alterations in cellular lipid homeostasis.

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

“…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] . For instance, OzID implemented with matrix-assisted laser desorption ionization (MALDI) MSI revealed altered fractions of phosphatidylcholine (PC) sn-isomers in a tumorous mouse brain 45 .…”

Positional determination of the carbon–carbon double bonds in unsaturated fatty acids mediated by solvent plasmatization using LC–MS

“…Determination of the positions and number of double bonds, as well as the chain length, is crucial for understanding the precise physiological functions of each FA species. Many methods have been established for the positional determination of double bonds by modifying the target FAs at the double bond positions or the terminal carboxyl group to yield diagnostic fragment ions [8][9][10][11][13][14][15][16][17][18][19][20][21][22][23][24][25][26] . CID-mediated fragmentation on ESI-MS or EI-mediated fragmentation on GC-MS of unsaturated FAs cannot always produce informative product ions to determine the double bond positions, especially in case of PUFAs with many double bonds.…”

“…The FA standards were dissolved in a solution containing two volumes of chloroform and one volume of methanol with 0.05% (w/v) of 2,6-di-t-butyl-p-cresol (butylated hydroxytoluene; Nacalai Tesque, Kyoto, Japan, #11421-92) and stored at − 20 °C until use. Other reagents used were: Deuterium oxide (D 2 O, FUJIFILM Wako Pure Chemical Corp., Osaka, Japan, #049-34242), Water- 18 O (H 2 18 O, Taiyo Nippon Sanso Corp., Tokyo, Japan, #F03-0027), high-performance liquid chromatography (HPLC)-grade tert-butyl methyl ether (t-BME, Sigma-Aldrich, #34875), HPLC-grade acetonitrile (FUJIFILM Wako, #019-08631), HPLC-grade acetone (Wako, #014-08681), HPLC-grade 1 mM ammonium acetate (FUJIFILM Wako, #018-21041), and 10% ammonia solution (FUJIFILM Wako, #013-17505).…”

“…The analytical mechanisms of this method were also studied. To elucidate the origin of oxygen in the epoxide and peroxide groups of the FAs formed by the plasma-ESI, we used 18 O and acetonitrile, and was examined by direct infusion assay (additives like ammonium acetate and ammonia solution were not supplemented to eliminate any contaminant of regular water). Epoxide formed by plasma-ESI had the same m/z values as those obtained using regular water (Fig.…”

“…Epoxidation of the double bond of FAs by low-temperature plasma successfully produces the informative diagnostic fragment ions from free FAs and lipid-conjugated FAs after CID fragmentation 15,16 . Epoxidation was also facilitated by use of catalytic agent [17][18][19] . Paper-spray ionization followed by CID also successfully epoxidize FAs and produce the same type of diagnostic fragment ions to identify the position of carbon-carbon double bonds 20 .…”