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. Lipid oxidation is a process inevitably connected to life. Although essential for all respiring organisms, oxygen can form reactive species (ROS). The accumulation of these compounds leads to the phenomenon commonly referred to as oxidative stress in microbes and plant and animal tissues. ROS play a role in the attack against microbial pathogens by host cells (Imlay, 2013), time-dependent death of dormant seeds (Lee et al., 2010), human diseases (Coyle and Puttfarcken, 1993;Giugliano et al., 1996), in aging (Sohal and Weindruch, 1996;Sohal et al., 2002), and in signaling processes (Gilroy et al., 2016;Mignolet-Spruyt et al., 2016) Unlike oxidized fatty acids (FAs), oxidized lipids are hardly available commercially. Targeted liquid chromatography-mass spectrometry (LC-MS) analysis of oxidized lipids using authentic standards is not very common (Hui et al., 2010;Ravandi et al., 2014). Often, oxidized lipids are detected using tandem mass spectrometry (MS/MS), where the collision-induced products of precursor ions (PRs) are scanned for fragments or neutral losses (NLs) that correspond to oxidized acyl residues expected in oxidized lipids (Spickett and Pitt, 2015), and in some cases, higher resolution MS/MS spectra are provided to support the identification (Buseman et al., 2006;Vu et al., 2012).High-resolution mass spectrometry (MS) allows for ...

Age‐dependent loss of seed viability is associated with increased lipid oxidation and hydrolysis

“…Using LC‐MS, we recorded untargeted lipid profiles of the wheat 1 set and identified nonoxidized and oxidized lipids by accurate mass and MS/MS fragmentation patterns (Table S3). For details, see Riewe et al (). Pseudospectra (composed by insource fragments, adducts, and isotopes from single analytes; Kuhl et al, ) for 8,836 nonredundant analytes were constructed by a deconvolution of all 18,556 quantitative m/z features of the dataset.…”

“…Approximately 50% (4,222) of all lipophilic analytes were correlated to germination, and, as with central metabolites, the number of negative correlations (2,878) was higher than the number of positive correlations (1,344), although a wider range ( R = −.89 to.81) was observed (Figure d, all correlation coefficients of known and unknown lipids are provided in Table S3). Of the 8,836 detected analytes, 771 were previously annotated as nonoxidized and oxidized (having one to four additional oxygen atoms) major lipids (Riewe et al, ). Ten annotations of (oxidized) (lyso)phosphatidic acids with lower annotation confidence were added from the negative mode recordings (Table S3) to complement this study.…”

“…Using LC-MS, we recorded untargeted lipid profiles of the wheat 1 set and identified nonoxidized and oxidized lipids by accurate mass and MS/MS fragmentation patterns (Table S3). For details, see Riewe et al (2017). Pseudospectra Table S3).…”

“…Pseudospectra Table S3). Of the 8,836 detected analytes, 771 were previously annotated as nonoxidized and oxidized (having one to four additional oxygen atoms) major lipids (Riewe et al, 2017). Ten annotations of (oxidized) (lyso)phosphatidic acids with lower annotation confidence were added from the negative mode recordings (Table S3) to complement this study.…”

“…Oxidized lipids from other major lipid classes were not reported in this study. However, hundreds of different singly and multiply oxidized lipids of various classes are formed in long-term stored wheat seeds (Riewe, Wiebach, & Altmann, 2017).…”