LC–MSⁿ Workflow Enhances High-Throughput Structural Analysis of Plant GIPCs for Glycosphingolipidomics

In a recent study published in Plant Journal (1), a research team from the University of Vienna explored a new method for high-throughput structural profiling of glycosyl inositol phospho ceramides (GIPCs) By integrating advanced liquid chromatography–mass spectrometry (LC–MS) with multistage fragmentation (MSⁿ) and automated annotation, the team enabled detailed characterization of GIPC series A–F.

Glycosyl inositol phospho ceramides (GIPCs)are the most abundant sphingolipids in plants, forming a major structural component of the plasma membrane in both dicotyledonous and monocotyledonous species. Despite their ubiquity, the structural complexity and functional diversity of GIPCs have made them analytically challenging to study. These lipids play essential roles in membrane organization, signal transduction, plant development, immune responses, and adaptation to environmental stress. GIPC biosynthesis occurs in the Golgi apparatus through a conserved pathway involving multiple glycosyltransferases.

in this second part of a two-part interview, LCGC International spoke with Evelyn Rampler of the Department of Analytical Chemistry at the University of Vienna and corresponding author of the aforementioned article to discuss how her team developed a robust LC–MS/MS platform for semiquantitative glycosphingolipidomics in barley, enabling detailed structural annotation of GIPCs across developmental stages and stress conditions, and paving the way for comparative analyses across plant species.

You used C16-lactosyl ceramide as an internal standard. What criteria led to its selection, and how did it support semiquantitative comparisons across development stages and heat stress conditions?

We selected C16-lactosyl ceramide as an internal standard based on several practical and analytical criteria. In a prior experiment, we confirmed that this compound is not endogenously present in plants using our RP-HRMSⁿ method, which makes it ideal for use as an internal reference. It is also commercially available, relatively inexpensive, and structurally similar to GIPC—containing two hexose units—making it well-suited to mimic GIPC behavior during extraction and ionization. By spiking it directly at the extraction step, we were able to correct for both extraction variability and instrument-related losses. This enabled a robust semiquantitative comparison of GIPC abundance across different barley developmental stages and under heat stress conditions, even in the absence of a commercial GIPC standard.

What measures did you implement to ensure chromatographic reproducibility and spectral stability across different LC–MS platforms and experimental conditions?

We analyzed a subset of samples using a different platform, the ZenoTOF 7600 system coupled to an Agilent Infinity 1290, which we also have available in our laboratory. The analysis of barley samples on this instrument led to the annotation of the same major GIPC species and the same elution order, with a retention time difference of around 1 min due to the different LC systems. Further, wine samples, which contain different GIPC classes (mostly A-series GIPC with an OH rest group), were also analyzed on both systems showing a similar inter-system reproducibility.

Hydrolysis was used to enrich GIPC over bulk lipids. How did the use of KOH enhance sample preparation, particularly in less developed barley grains?

Based on established literature, we applied alkaline hydrolysis with 0.1 M KOH in methanol to remove bulk lipids such as glycerophospholipids. We observed that it also disrupted the starchy matrix—especially in less developed barley grains—reducing desludging and gelatinization. This improved both sample preparation and GIPC recovery, enabling reliable, high-throughput LC–MS analysis across different barley samples.

How did negative ion mode MS² and MS³ contribute to resolving sugar stereochemistry or branching in higher GIPC series—especially given the lack of standards?

Negative ion mode MS² and MS³ spectra were key for detecting glycan branching in higher GIPC series. Instead of the IP fragments at m/z 259 and 241, we observed new diagnostic ions at m/z 403 and 421, indicating that a hexose was directly linked to the inositol ring—evidence of branching. These insights, integrated into our decision rules in LDA, enabled automated annotation of complex GIPC, even without commercial standards.

What challenges remain in stereochemical resolution of GIPC glycan moieties, and what analytical innovations could help overcome these in future studies?

Significant challenges remain in the analysis of intact glycosphingolipids, especially in resolving glycan and lipid isomers. With current workflows, we can’t distinguish between sugars like glucose, mannose, or galactose—only generic losses such as hexose or HexNAc. On the ceramide side, we can now determine the number of hydroxylations and double bonds, but not their exact positions, which would require more advanced fragmentation techniques such as electron-activated dissociation (EAD). Our current goal is to develop a global glycosphingolipidomics platform, including additional acidic and neutral GSL classes. However, this is still challenging due to the lack of commercial standards and automated annotation workflows. Advances in novel separation methods—such as ion mobility spectrometry (IMS), supercritical fluid chromatography (SFC), HILIC, or 2D-LC—could improve glycan and lipid isomer separation. Furthermore, next-generation high-resolution mass spectrometers, such as the ZenoTOF 8600 (Sciex) or Orbitrap Astral Zoom (Thermo), combined with enhanced fragmentation capabilities and automated annotation solutions, will be essential to push the field of glycosphingolipidomics to the next level. Ultimately, these analytical advancements must be applied to meaningful biological questions to fully realize their potential.

Given the demonstrated species-specific and developmental variation in GIPC profiles, how could this LC–MS/MS platform aid in comparative glycosphingolipidomics across plant species or tissue types?

Our presented RP-HRMSⁿ workflow is well-suited for advancing comparative glycosphingolipidomics by enabling high-resolution structural annotation of GIPCs—including glycan branching, series types (A–D), and ceramide features like chain length and hydroxylation. While we demonstrated developmental and heat stress-induced remodeling of GIPCs in whole barley grains, the logical next step is to apply this approach to tissue-specific separation and other plant species. This would allow us to explore the still poorly understood biological roles of GIPCs in more detail.

By separating tissues or subcellular compartments—like the endoplasmic reticulum (ER), plasma membrane, or vesicles—we could uncover their involvement in membrane dynamics, intracellular trafficking, or ER-specific stress responses. Similarly, profiling GIPCs in pathogen-challenged tissues could shed light on their role in host–pathogen interactions, especially considering their known link to NLP toxin binding. The method can also be extended to study GIPC patterns across developmental stages, plant diseases, and abiotic stresses such as heat or drought. Thanks to decision rule-based annotation, it’s ideal for identifying potential GIPC-based markers of stress or development, paving the way for new insights into plant lipid function and crop resilience. In summary, by combining detailed structural analysis, automated annotation, and tissue-level resolution, this platform offers a powerful tool for uncovering the functional diversity and localization of GIPCs across species and biological contexts.

References

1. Pühringer, M.; Thür, N.; Schnurer, M. et al. 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. Plant J. 2025, 122 (6), e70279. DOI: 10.1111/tpj.70279