Comprehensive and quantitative analysis of lysophospholipid molecular species present in obese mouse liver by shotgun lipidomics

Abstract

Shotgun lipidomics exploits the unique chemical and physical properties of lipid classes and individual molecular species to facilitate the high-throughput analysis of a cellular lipidome on a large scale directly from the extracts of biological samples. A platform for comprehensive analysis of lysophospholipid (LPL) species based on shotgun lipidomics has not been established. Herein, after extensive characterization of the fragmentation pattern of individual LPL class and optimization of all experimental conditions including developing new methods for optimization of collision energy, and recovery and enrichment of LPL classes from the aqueous phase after solvent extraction, a new method for comprehensive and quantitative analysis of LPL species was developed. This newly developed method was applied for comprehensive analysis of LPL species present in mouse liver samples. Remarkably, the study revealed significant accumulation of LPL species in the liver of ob/ob mice. Taken together, by exploiting the principles of shotgun lipidomics in combination with a novel strategy of sample preparation, LPL species present in biological samples can be determined by the established method. We believe that this development is significant and useful for understanding the pathways of phospholipid metabolism and for elucidating the role of LPL species in signal transduction and other biological functions.

Figure 1

Elucidation of fragmentation patterns of lysophospholipid classes with representative synthetic species. Product ion ESI-MS analyses of synthetic 16:0 lysophosphatidylinositol (LPI) (A), 17:1 LPI (B), 16:0 lysophosphatidylserine (LPS) (C), and 17:1 LPS (D) at collision pressure of 1 mT and collision energy of 35 and 22 eV for LPI and LPS species, respectively, were performed as described under “MATERIALS AND METHODS”. Tandem MS analysis was performed to identify the common fragmentation patterns of LPI (A and B) and LPS (C and D), which were used for identification and quantification of these LPL species based on the principles of multi-dimensional mass spectrometry-based shotgun lipidomics. FA denotes fatty acyl (or fatty acid).

Figure 2

Demonstration of optimizing collision energy for quantification of lysophospholipid (LPL) species. A series of solutions containing an identical amount of lipid extract from rat liver, but varied amounts of 17:1 lysophosphatidylserine (LPS) as an internal standard (IS) for quantification of LPS species were prepared. These solutions were subject to tandem MS analyses of neutral loss of 87 Da (NLS87) at different collision energy as described under “MATERIALS AND METHODS”. Plots of I_S/I_X acquired from NLS87 vs. C_S/C_X at CE of 15 (diamonds), 20 (squares), and 25 (circles) eV were illustrated for LPS species (X) of 18:0 (A), 18:1 (B), 20:4 (C), and 22:6 (D), where I_S was the peak intensity of IS (i.e., 17:1 LPS) at the concentration of C_S; whereas I_X was the peak intensity of species X which was present in rat liver extract and estimated as described under “MATERIALS AND METHODS”. Since I_X was virtually constant under the experimental conditions, the slopes of the plots only depended on the values of CE. The values of these slopes were used to determine the optimized CE value for individual MS/MS scan mode as described under “MATERIALS AND METHODS”. The optimized CE value for individual MS/MS scan mode was summarized in Table 1.

Figure 3

Representative determination of linear dynamic range of the method with optimized collision energy for quantification of lysophosphatidylserine (LPS) species. A fixed amount of lipid extract from rat liver was added with different amount of 17:1 LPS as an internal standard (IS) (28 pmol/mg protein, panel A; 112 pmol/mg protein, panel B; 224 pmol/mg protein, panel C; and 672 pmol/mg protein, panel D) for quantification of LPS species present in rat liver extracts. Linear regression (panel E) of peak intensity ratios (I_X/I_IS) of liver LPS species (X) and IS vs. their molar ratio (C_X/C_IS) was performed and the linear result was plotted as its logarithm format to demonstrate the true linearity of the data as previously described.

Figure 4

Representative MS/MS measurement of the content of lysophospholipid (LPL) species in lipid extracts from rat liver. Representative MS/MS spectra of NLS87 (lysophosphatidylserine, LPS, panel A), PIS153 (lysophosphatidic acid, LPA, panel B), NLS316 (lysophosphatidylinositol, LPI, panel C), NLS228 (lysophosphatidylglycerol, LPG, panel D), NLS59 (lysophosphatidylcholine, LPC, panel E), and NLS43 (lysophosphatidylethanolamine, LPE, panel F) were acquired from lipid extracts of rat liver samples as described under “MATERIALS AND METHODS” with the experimental settings summarized in Table 1. NLS, PIS, and IS stand for neutral loss scan, precursor-ion scan, and internal standard, respectively.

Figure 5

Comparison of the content of individual lysophospholipid (LPL) species in lipid extracts from wild-type vs. ob/ob mouse liver samples. The content of individual LPL species in lipid extracts of wild-type (open bars) vs. ob/ob (solid bars) mouse liver samples was determined by tandem MS analysis as illustrated in Figure 4, each of which was obtained by ratiometric comparison of individual ion peak intensity with that of internal standard (IS) of the class. LPI, LPG, LPC, LPE, LPA, and LPS denote lysophosphatidylinositol, lysophosphatidylglycerol, lyso choline glycerophospholipid, lysophosphatidylethanolamine, lysophosphatidic acid, and lysophosphatidylserine, respectively. The prefix “a” and “p” stand for plasmanyl- and plasmenyl-containing species, respectively. All data are presented as the means ± SD of four different animals. Statistical significance was determined by a two-tailed student t-test in comparison to control, where *p < 0.05, **p < 0.01, and ***p < 0.001.