Overview of Lipidomic Analysis of Triglyceride Molecular Species in Biological Lipid Extracts

Abstract

Triglyceride (TG) is a class of neutral lipids, which functions as an energy storage depot and is important for cellular growth, metabolism, and function. The composition and content of TG molecular species are crucial factors for nutritional aspects in food chemistry and are directly associated with several diseases, including atherosclerosis, diabetes, obesity, stroke, etc. As a result of the complexities of aliphatic moieties and their different connections/locations to the glycerol backbone in TG molecules, accurate identification of individual TG molecular species and quantitative assessment of TG composition and content are particularly challenging, even at the current stage of lipidomics development. Herein, methods developed for analysis of TG species, such as liquid chromatography-mass spectrometry with a variety of columns and different mass spectrometric techniques, shotgun lipidomics approaches, and ion-mobility-based analysis, are reviewed. Moreover, the potential limitations of the methods are discussed. It is our sincere hope that the overviews and discussions can provide some insights for researchers to select an appropriate approach for TG analysis and can serve as the basis for those who would like to establish a methodology for TG analysis or develop a new method when novel tools become available. Biologically accurate analysis of TG species with an enabling method should lead us toward improving the nutritional quality, revealing the effects of TG on diseases, and uncovering the underlying biochemical mechanisms related to these diseases.

Keywords: lipidomics; mass spectrometry; metabolic syndrome; regioisomers; shotgun lipidomics; triglycerides.

Figure 1.

Schematic illustration of TG isomers yielded from different numbers of fatty acyls of X, Y, and Z. The regioisomers due to different locations of fatty acids are highlighted with red and the enantiomers from their left side are highlighted with green.

Figure 2.

Silver-ion HPLC/APCI-MS chromatogram of the randomization mixture prepared from triolein (OOO, Δ9-C18:1), trilinolenin (LnLnLn, Δ9,12,15-C18:3) and tri-γ-linolenin (γLnγLnγLn, Δ6,9,12-C18:3). Numbers correspond to the double bond number. (Modified from ref. with permission from Elsevier, Copyright 2010)

Figure 3.

Identification of TG species in soybean lipid extract using SFC-MS. The 2D map shows a magnified view of SFC-MS data obtained by tandem three Chromolith Performance RP-18e columns. Small circle: peak top of each TG species. There are two types of groups that have the pattern of TG arrangement as indicated with arrows. Line arrows: a group of TG species with an sn-1 fatty acyl changed (box), e.g., OLP, LLP, and LnLP (A). Dotted arrows: a group of TG species with an sn-2 fatty acyl changed (circle), e.g., POP, PLP, and PLnP (B). (Modified from ref. with permission from Elsevier, Copyright 2011).

Figure 4.

Representative tandem MS analysis of lithium TG adducts in the product-ion mode. Tandem MS spectra of lithiated 16:0–20:4–18:1 (Panel A) and 18:1–20:4–18:1 TG species (Panel B) were obtained using a ThermoFisher TSQ Altis mass spectrometer with collision energy of 32 eV and collision gas pressure of 1 mTorr. The tandem MS spectral analyses demonstrated the abundant fragment ions yielded from neutral losses of either free fatty acids or lithium fatty acyl salts from corresponding TG lithium adducts.

Figure 5.

A full mass spectrum and total ion current chromatogram of stepwise acquisition of neutral loss scans for identification and quantitation of TG molecular species in lipid extracts of rat superior mesenteric ganglia. The lipid extract from rat superior mesenteric ganglia was analyzed in the positive-ion mode after infusion of the diluted lipid extract in the presence of a small amount lithium chloride directly with a NanoMate device. A positive-ion full mass spectrum of lipid extracts of rat superior mesenteric ganglia was acquired in a survey scan mode by a QqQ-type mass spectrometer (Thermo Fisher Scientific TSQ Altis) (Panel A). The stepwise acquisition of neutral loss (NL) scans as indicated (Panel B) was conducted using a sequential and customized program operating under Xcalibur software. Each segment of individual NL scan was taken for 2 min in the profile mode. Panel C shows an example of NL scan averaged from all of scans acquired in the segment corresponding to NL310 (i.e., 20:1 fatty acid) which is usually present in very low abundance.

Figure 6.

A representative two-dimensional ESI/MS analysis of TG molecular species in rat superior mesenteric ganglion lipid extracts. The full mass spectrum acquired in the survey scan mode (the most top trace, the first dimension of the two-dimension mapping) was obtained in the positive-ion mode after direct infusion with a NanoMate device. Neutral loss (NL) scans of all naturally-occurring aliphatic chains of lipid extracts of rat superior mesenteric ganglia, serving as building blocks were acquired and utilized to identify TG molecular species, deconvolute isomeric molecular species, and quantify TG individual molecular species by comparisons with a selected internal standard. Each MS or MS/MS scan of two-dimensional ESI mass mapping was acquired by sequentially programmed custom scans operating under Xcalibur software as shown in Figure 5. For tandem mass spectrometry in the positive-ion neutral loss (NL) mode, both the first and third quadrupoles were coordinately scanned with a mass difference (i.e., neutral loss) corresponding to the neutral loss of a non-esterified fatty acid from TG molecular species, while collisional activation was performed in the second quadrupole. All mass spectral traces were displayed after normalization to the base peak in individual scan.

Figure 7.

Schematic illustration of fragmentation pathways yielding sn-2 vs sn-3 fatty acyl losses from a lithiated TG ion. Panels A and B indicate both sn-1 and -3 fatty acyls can provide the alpha-hydrogen atom to facilitate the sn-2 fatty acyl salt elimination. Panel C indicates only sn-2 fatty acyl can provide the alpha-hydrogen atom to facilitate either sn-1 (not shown) or sn-3 fatty acyl salt elimination. R_n = R_n’ = R_n”. (Modified from ref. with permission from Elsevier, Copyright 1999).

Figure 8.

Schematic illustration of a TG de novo synthesis model for simulation of TG ion composition quantified using the MDMS-SL technology. TG molecules are biosynthesized with reacylation of diglyceride (DG) molecules of various sources yielded mainly through dephosphorylation of phosphatidic acid (PA) (DG_PA), reacylation of monoglyceride (MG) (DG_MG), and, to a less extent (as indicated with a broken line arrow), through hydrolysis of phosphatidylinositol (PI) with phospholipase C (PLC) activities (DG_PI). The levels of the metabolic pathways contributing to the TG content were calculated via simulation of each TG ion profile including all neutral loss scans with parameters of K₁, K₂, and K₃ using the equations (1) to (3) with the restriction of equation (4), respectively. These parameters represent the probabilities of each DG source being reacylated to TG. In addition, the parameters of k₁, k₂, and k₃ were used in the sn-1, 2, and 3 reacylation steps of TG species in the forms of exp(−k₁.x_j), exp(k₂.x_j), and exp(−k₃.x_j), respectively, where k₁ and k₃ represented a simulated decay constant whereas k₂ represented a simulated enhancing constant, and x_j is the number of double bonds present in the corresponding fatty acyl chain. MGAT and DGAT stand for MG and DG acyltransferases, respectively. The multiple arrows at the k₂ step indicate that MG molecules could be produced from various sources including lysoPA dephosphatation, DG hydrolysis, and glycerol acylation. (Modified from ref. with permission from the American Society for biochemistry and molecular biology, Copyright 2013). Lipase-mediated hydrolysis of TG species is not considered under steady state conditions as discussed in the text.