Serum lipidomics profiling using LC-MS and high-energy collisional dissociation fragmentation: focus on triglyceride detection and characterization

Abstract

There is a growing need both clinically and experimentally to improve the characterization of blood lipids. A liquid chromatography-mass spectrometry (LC-MS) method, developed for the qualitative and semiquantitative detection of lipids in biological samples and previously validated in mitochondrial samples, was now evaluated for the profiling of serum lipids. Data were acquired using high-resolution, full scan MS and high-energy, collisional dissociation (HCD), all ion fragmentation. The method was designed for efficient separation and detection in both positive and negative ionization mode and evaluated using standards spanning seven lipid classes. Platform performance, related to the identification and characterization of serum triglycerides (TGs), was assessed using extracted ion chromatograms with mass tolerance windows of 5 ppm or less from full scan exact mass measurements determined using SIEVE nondifferential LC-MS analysis software. The platform showed retention time coefficients of variation (CV) of <0.3%, mass accuracy values of <2 ppm error, and peak area CV of <13%, with the majority of that error coming from sample preparation and extraction rather than the LC-MS analysis, and linearity was shown to be over 4 orders of magnitude (r(2) = 0.999) for the standard TG (15:0)(3) spiked into serum. Instrument mass accuracy and precision were critical to the identification of unknown TG species, in part because these parameters enabled us to reduce false positives. In addition to detection and relative quantitation of TGs in serum, TG structures were characterized through the use of alternating HCD scans at different energies to produce diagnostic fragmentations on all ions in the analysis. The lipidomics method was applied to serum samples from 192 rats maintained on diets differing in macronutrient composition. The analysis identified 86 TG species with 81 unique masses that varied over 3.5 orders of magnitude and showed diet-dependency, consistent with TGs linking diet and disease risk.

Figure 1

Panels A and B show the total ion chromatogram (TIC) separation of the same serum pool sample using LC-MS performed in negative and positive ionization modes, respectively. Sections of the chromatograms are labeled with the lipid categories detected, indicating the regions where each will elute using the LC-MS method. Highlighted in italics are some lipid classes from those categories that are observed in the serum pool samples. A more detailed list is in Table 1.

Figure 2

shows the two possible structures of TG (50:0) found in the HMDB, [M+NH4] + m/z 852.8015, as a. and b. of both panels 1 and 2. Panel 1 indicates HCD fragmentation at 30 eV to the unique and shared diacylglycerol fragments on each structure and panel 2 shows the FA fragments from HCD fragmentation at 60 eV.

Figure 3

shows the XIC of parent ion m/z 852.8015 in panel A, with panels B–D showing XICs of the 3 possible 30 eV diacylglycerol fragments shown in Figure 2. Chromatographic alignment of the parent ion peak at 24.05 minutes can be seen with fragments m/z 579 and m/z 551 in panels C and D.

Figure 4

the top panel, shows the full mass spectrum of the species with an RT of 24.05 minutes from the chromatogram in Figure 3, indicating the parent ion peak of interest at m/z 852.8010. The bottom panel shows the all ion fragmentation mass spectrum at RT 24.06 minutes between m/z 530 - m/z 620. The fragmentations resulting from the parent ion are labeled at m/z 551 and m/z 579, with fragment m/z 607 clearly marked as an M+2 isotope of m/z 605, not a unique fragment from the parent ion of interest.

Figure 5

shows the XIC of parent ion m/z 852.8015 in panel A, with panels B–D showing XICs of the 3 possible 60 eV FA fragments shown in Figure 2. Chromatographic alignment of the parent ion peak at 24.05 minutes can be seen with fragments m/z 239 and m/z 267 in panels C and D, confirming the unknown TG (50:0) to be TG (16:0/16:0/18:0).

Figure 6

shows the bar graphs representing the amount of TG (16:0/16:0/18:1) or TG (16:0/18:1/18:0) (panel A and B respectively) found in serum samples of animals held on each fat diet. In this analysis, the 4 carbohydrate diets did not affect the amount of TGs found; the trends observed were solely dictated by the major fat constituent consumed. Bars show means +/− s.e.m. (standard error of the mean)