Structure Annotation and Quantification of Wheat Seed Oxidized Lipids by High-Resolution LC-MS/MS

Abstract

Lipid oxidation is a process ubiquitous in life, but the direct and comprehensive analysis of oxidized lipids has been limited by available analytical methods. We applied high-resolution liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) to quantify oxidized lipids (glycerides, fatty acids, phospholipids, lysophospholipids, and galactolipids) and implemented a platform-independent high-throughput-amenable analysis pipeline for the high-confidence annotation and acyl composition analysis of oxidized lipids. Lipid contents of 90 different naturally aged wheat (Triticum aestivum) seed stocks were quantified in an untargeted high-resolution LC-MS experiment, resulting in 18,556 quantitative mass-to-charge ratio features. In a posthoc liquid chromatography-tandem mass spectrometry experiment, high-resolution MS/MS spectra (5 mD accuracy) were recorded for 8,957 out of 12,080 putatively monoisotopic features of the LC-MS data set. A total of 353 nonoxidized and 559 oxidized lipids with up to four additional oxygen atoms were annotated based on the accurate mass recordings (1.5 ppm tolerance) of the LC-MS data set and filtering procedures. MS/MS spectra available for 828 of these annotations were analyzed by translating experimentally known fragmentation rules of lipids into the fragmentation of oxidized lipids. This led to the identification of 259 nonoxidized and 365 oxidized lipids by both accurate mass and MS/MS spectra and to the determination of acyl compositions for 221 nonoxidized and 295 oxidized lipids. Analysis of 15-year aged wheat seeds revealed increased lipid oxidation and hydrolysis in seeds stored in ambient versus cold conditions.

Figure 1.

LC-MS analysis in positive mode. A, Example base peak chromatogram. B, Median retention time, m/z, and intensity (point size corresponds to log₁₀₀₀ transformed median ion count) of 18,556 chromatographic m/z features in 90 wheat seed stocks. Six hundred twenty-four identified lipids (with MS/MS validation) and 157 FAs (without MS/MS validation) are displayed in colors representing their lipid class. The boxed area is shown in higher magnification in C. C, O₁ (red) TAGs elute 40 s earlier than their cognate O₀ TAGs (black). The boxed area is shown in higher magnification in D. D, Higher oxidized TAGs occur as isomers with different retention times.

Figure 2.

Examples of MS/MS validation and acyl composition determination for lipids of eight different classes. NLs and fragments (FR) of FAs and head groups were computationally identified and used to validate the PR annotation for O₄ TAG 54:7 (Supplemental Table S1, Formula_ID = 9409; for the chromatographic profile, see Fig. 1D [A]), O₁ DAG 36:5 (Formula_ID = 7380 [B]), O₁ MAG 18:3 (Formula_ID = 1302 [C]), O₃ PC 36:4 (Formula_ID = 2322 [D]), O₂ lysoPC 18:2 (Formula_ID = 1850 [E]), O₁ PE 36:4 (Formula_ID = 1440 [F]), O₂ MGDG 36:3 (Formula_ID = 8269 [G]), and O₂ DGDG 34:2 (Formula_ID = 8128 [H]). Only subsets of the indicated fragments are depicted. Multiple occurrences of the same FA within the different spectra are indicated by identical colors. FAs are labeled in purple, blue, or red, and head groups are labeled in green. Annotations in black were not obtained computationally and are only used to provide additional information in the example spectra. Representation of the acyl combination in the MS/MS spectra is expressed as cumulative abundance of all explanatory fragments/NLs in percentage of the base peak.

Figure 3.

Quantitative analysis of oxidized lipids in long-term stored wheat seeds under ambient and cold conditions. A to M, For each of the 13 detected lipid classes, the number of detected lipid species is given (number in parentheses) and broken down into nonoxidized (nO₀) and oxidized (nO_1-4) lipids. Peak areas of all nonoxidized and oxidized lipids were summed up for each of the five seed stocks stored in the cold (CS) or at ambient temperature (AS) from 1998 to 2013. N, TAGs were reanalyzed from 1:10 diluted extracts. Data are means ± se (n = 5), and significant differences between AS and CS are indicated by ANOVA P values.