Chasing the Major Sphingolipids on Earth: Automated Annotation of Plant Glycosyl Inositol Phospho Ceramides by Glycolipidomics

Abstract

Glycosyl inositol phospho ceramides (GIPCs) are the major sphingolipids on earth, as they account for a considerable fraction of the total lipids in plants and fungi, which in turn represent a large portion of the biomass on earth. Despite their obvious importance, GIPC analysis remains challenging due to the lack of commercial standards and automated annotation software. In this work, we introduce a novel GIPC glycolipidomics workflow based on reversed-phase ultra-high pressure liquid chromatography coupled to high-resolution mass spectrometry. For the first time, automated GIPC assignment was performed using the open-source software Lipid Data Analyzer (LDA), based on platform-independent decision rules. Four different plant samples (salad, spinach, raspberry, and strawberry) were analyzed and the results revealed 64 GIPCs based on accurate mass, characteristic MS2 fragments and matching retention times. Relative quantification using lactosyl ceramide for internal standardization revealed GIPC t18:1/h24:0 as the most abundant species in all plants. Depending on the plant sample, GIPCs contained mainly amine, N-acetylamine or hydroxyl residues. Most GIPCs revealed a Hex-HexA-IPC core and contained a ceramide part with a trihydroxylated t18:0 or a t18:1 long chain base and hydroxylated fatty acid chains ranging from 16 to 26 carbon atoms in length (h16:0–h26:0). Interestingly, four GIPCs containing t18:2 were observed in the raspberry sample, which was not reported so far. The presented workflow supports the characterization of different plant samples by automatic GIPC assignment, potentially leading to the identification of new GIPCs. For the first time, automated high-throughput profiling of these complex glycolipids is possible by liquid chromatography-high-resolution tandem mass spectrometry and subsequent automated glycolipid annotation based on decision rules.

Decoding Distinct Ganglioside Patterns of Native and Differentiated Mesenchymal Stem Cells by a Novel Glycolipidomics Profiling Strategy

“…We already demonstrated that different glycolipid classes can be added using the presented glycolipidomics annotation, e.g., for glycosyl inositol phospho ceramides in plants. 59 Hence, our LDA-based annotation strategy is currently the only automated glycolipid annotation software available for the community. Overall, our proposed workflow can be seen as the hallmark for shedding light on the enigmatic processes of gangliosides and glycolipids in general, which are critical mediators in cell−cell interaction and signaling pathways.…”

“…Our presented open-source annotation workflow based on LDA can be easily applied to any other hyphenated liquid chromatography and MS setup by including new and/or adapting existing decision rules reflecting fragmentation information obtained by (ideally iso-pure) glycolipid standards. We already demonstrated that different glycolipid classes can be added using the presented glycolipidomics annotation, e.g., for glycosyl inositol phospho ceramides in plants . Hence, our LDA-based annotation strategy is currently the only automated glycolipid annotation software available for the community.…”

Recurrent Topics in Mass Spectrometry-Based Metabolomics and Lipidomics—Standardization, Coverage, and Throughput

“…and decision rule-based identification (LDA, LipidXplorer, LipidMatch, LipidHunter, etc.). Due to building blocks of lipids leading to a distinct MS2 pattern within the same class, decision rule sets based on well-defined fragments (fragment rules) and their intensity relationships (intensity rules) can be described for specific lipid classes. , For library matching similar principles are applied as in standard metabolomics workflows using accurate mass, MS2 spectra, and scoring algorithm. Both experimental or in-silico databases are applied in lipidomics.…”

Sphingolipid metabolism, transport, and functions in plants: Recent progress and future perspectives

“…A high-performance liquid chromatography (HPLC) analysis identified the major plant C18 LCBs, including t18:1(Z), t18:1(E), t18:0, d18:1(Z), d18:1(E), and d18:0 ( Chen et al., 2006 ). Plants also contain traces of another four C18 LCBs (d18:2(4E,8E), d18:2 (4E,8Z), t18:2 [with unknown structure], and a unique d18:1 [with a C10=C11 double bond]), one C20 LCB t20:0, and special d21:1 or d22:1 LCBs (the last two LCBs contain a C7=C8 double bond), which have been validated by either mass spectrometry (MS) or nuclear magnetic resonance (NMR) ( Kimberlin et al., 2013 ; Ngo et al., 2020 ; Panzenboeck et al., 2020 ; Wang et al., 2020a ). However, the two common LCBs d20:0 and d20:1, which are detected in animals and Saccharomyces cerevisiae , are rarely reported in plant samples ( De Castro Levatti et al., 2017 ; Zhu et al., 2020 ).…”

“…canaense V.S. Dang IR, UV, HR-ESI-MS, HR-ESI-MS/MS and NMR Panzenboeck et al., 2020 Found four GIPCs containing t18:2 in the Rubus idaeus sample Lactuca sativa var . capitata nidus tenerimma , Spinacia oleracea , Rubus idaeus and Fragaria RP-HRMS/MS using the open-source program LDA for automated GIPC assignment ESI/MS, electrospray ionization MS; ESI-MS/MS, electrospray ionization tandem MS; MS; HPLC/ESI-MS/MS, high-performance liquid chromatography electrospray ionization tandem MS; GC/MS, gas chromatography-MS; MALDI-(Q)-TOF-MS, matrix-assisted laser desorption ionization (quadrupole) time-of-flight MS; UPLC-MS, ultraperformance liquid chromatography-tandem MS; IR, infrared spectroscopy; UV, ultraviolet; HPAEC-PAD, high-performance anion-exchange chromatography with pulsed amperometric detection; HR-ESI-MS/MS, high-resolution electrospray ionization tandem MS; LDA, an open-source software lipid data analyzer.…”