Automated mass spectrometry-based profiling of multi-glycosylated glycosyl inositol phospho ceramides (GIPC) reveals specific series GIPC rearrangements during barley grain development and heat stress response

Glycosyl inositol phospho ceramides (GIPC) are the predominant glycosphingolipids in plant membranes, essential for their membrane stability, cell signaling, stress adaptation, and pathogen resistance. However, their complex structures, characterized by a ceramide backbone and a glycan head group, have challenged comprehensive analysis using traditional methods, which often rely on separate glycan or lipid profiling. To overcome these limitations, we developed a glycosphingolipidomics assay using reversed-phase high-resolution mass spectrometry including multistage fragmentation (RP-HRMSⁿ). This method enables direct, detailed structural characterization of GIPC in plants, combining advanced chromatographic separation, multistage fragmentation, and automated annotation using decision rule-based criteria. Applied to barley grains, the assay identified 102 GIPC species, including A-, B-, C-, and D-series GIPC, previously unreported glycan branching fragments (421 and 403 m/z), and a huge structural variety in the ceramide moiety. Profiling at different development stages revealed dynamic GIPC regulation during grain development, with an upregulation of B- and C-series towards mature development stages. The application of heat stress induced significant remodeling of GIPC profiles, mainly through upregulation of B-series species, which emphasizes their roles in maintaining membrane stability and functionality under abiotic stress conditions. The presented glycosphingolipidomics assay enables the first automated analysis of complex GIPC through a decision rule-based identification approach. By resolving GIPC to the molecular lipid species level, the method provides novel insights into GIPC diversity, homeostasis, and their critical roles in membrane dynamics, stress adaptation, and pathogen resistance, paving the way for advanced research in plant lipidomics and stress biology.