MALDI FTMS Imaging Mass Spectrometry of N-glycans as Tissue Biomarkers of Cancer

Importance of the Topic

N-glycosylation is a widespread post-translational modification critical for protein folding, stability and cell–cell interactions. Alterations in N-glycan structures are intimately linked to cancer progression, influencing immune responses, extracellular matrix remodeling and tumor–stroma communication. Imaging N-glycans in formalin-fixed paraffin-embedded (FFPE) tissues unlocks valuable biomarker information from archived clinical specimens, supporting improved diagnosis, prognosis and therapeutic targeting.

Objectives and Study Overview

This study presents a complete workflow for spatial mapping of N-glycans in FFPE thyroid cancer sections using matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI FTMS) on a 7 tesla solariX platform. Focusing on anaplastic thyroid carcinoma, the aim was to detect, identify and quantify diverse glycoforms across tumor, necrotic and adjacent non-tumor regions, demonstrating the method’s sensitivity and specificity.

Methodology

A 5 µm FFPE section of anaplastic thyroid cancer was deparaffinized, subjected to acidic antigen retrieval, then coated with PNGase F via an automated sprayer. Following in situ enzymatic release of N-glycans at 37 °C under humid conditions, α-cyano-4-hydroxycinnamic acid matrix was applied. Imaging was performed in positive ion mode with 150 µm pixel spacing, 200 laser shots per pixel, spanning m/z 500–5,000. A transient length of 1.2 s achieved resolving power ~85 000 at m/z 1 850. Data processing involved FlexImaging for peak mapping, GlycoWorkbench for glycan annotation within 5 ppm tolerance, and SCiLS Lab segmentation with bisecting k-means and Manhattan metric. Statistical filtering used Wilcoxon rank sum (p≤1×10⁻³) and ROC analysis (AUC≤0.8).

Used Instrumentation

• TM-Sprayer (HTX Imaging) for enzyme/matrix deposition
• 7 T solariX MALDI FTMS with dual ESI/MALDI source
• Smartbeam II laser (minimum focus setting)
• FlexImaging software v4.1 (Bruker Daltonics)
• GlycoWorkbench 2.0 for glycan identification
• SCiLS Lab software 2016b (v4.01.8705) for segmentation and statistics

Main Results and Discussion

Over 111 distinct N-glycan species were detected as sodium adducts, including high-mannose, hybrid, complex and sialylated structures. The solariX’s cooled source preserved labile sialic acids, yielding equivalent sensitivity for sialylated and non-sialylated glycans. The workflow spanned m/z 1 257.4 (Man5) to m/z 4 000.4 (fucosylated tetraantennary polyLacNAc). SCiLS segmentation correlated glycan distributions with pathologist-annotated regions, revealing:

• Elevated Man9 in adjacent non-tumor tissue vs. anaplastic regions
• Biantennary structures predominating in necrotic zones
• Progressive increase of fucosylated tetraantennary glycans in tumor and necrosis

These spatially resolved signatures highlight the heterogeneity of the tumor microenvironment and underscore the capability of MALDI FTMS imaging for glycomic profiling.

Benefits and Practical Application

• Enables high-throughput screening of archived FFPE biobanks for glycan biomarkers
• Delivers spatially resolved molecular data without tissue destruction
• Supports discovery of glycosylation-based diagnostic and prognostic markers
• Facilitates investigation of glycan-mediated mechanisms in cancer biology

Future Trends and Opportunities

• Integration with multi-omics and imaging modalities (e.g., immunohistochemistry, spatial transcriptomics)
• Refinement of spatial resolution toward single-cell glycomics
• Targeted labeling of glycan epitopes to enhance specificity
• Application to other disease models (neurodegeneration, infectious diseases)
• Incorporation of machine learning for automated glycan pattern recognition

Conclusion

The described MALDI FTMS imaging workflow on a 7 T solariX provides a robust, sensitive and high-resolution approach for mapping N-glycans in FFPE tumor tissues. Preservation of sialic acids and broad m/z coverage enable comprehensive profiling of glycan alterations within the tumor microenvironment. This platform offers a powerful tool for glycomics research and biomarker discovery in cancer and beyond.

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