Integrated MALDI-MSI and Glycopeptide LC-MS/MS Reveal Site-and Region-Specific Glycosylation Changes in Tumor Tissue

Significance of the topic

Glycosylation is crucial for protein folding, cell signaling and immune responses. Alterations in N-glycan patterns often accompany malignant transformation, making spatial glycomic mapping a valuable tool in cancer research.

Objectives and Study Overview

This study aimed to combine high-resolution MALDI mass spectrometry imaging (MSI) of released N-glycans with LC-MS/MS analysis of intact glycopeptides to identify site-specific and region-specific glycosylation changes in tumor versus normal prostate tissue.

Methodology and Instrumentation

Adjacent sections of prostate tissue underwent parallel processing: released glycans were imaged by MALDI-MSI after antigen retrieval, PNGase F digestion and matrix application; glycopeptides were enriched by HILIC and analyzed by LC-MS/MS using GlycoPASEF on a timsTOF fleX platform. Data processing included spatial mapping of glycan classes and peptide-level glycoprotein identification.

Main Results and Discussion

The imaging data revealed enrichment of high-mannose, hybrid and bisected glycans in prostate cancer regions, while normal tissue showed predominance of complex-type fucosylated and sialylated structures. LC-MS/MS validated these spatial trends and detected differential expression of glycoproteins bearing the shared glycan motif H3N2F1. Notably, Galectin-3-binding protein carried this glycan in tumor samples, underscoring functional implications in tumor progression and immune modulation.

Benefits and Practical Applications

Integrating MSI with glycoproteomics provides comprehensive insights into glycosylation remodeling, facilitating discovery of candidate biomarkers and potential therapeutic targets in oncology.

Used Instrumentation

• MALDI-MSI on timsTOF fleX
• HILIC-based glycopeptide enrichment
• GlycoPASEF LC-MS/MS
• PNGase F digestion and standard sample preparation

Future Trends and Potential Applications

Advances may include higher spatial resolution imaging, deeper glycoproteome coverage, integration with other omics workflows and development of clinical glycoimaging platforms for diagnostics and therapeutic monitoring.

Conclusion

This dual-modality approach reveals site- and region-specific glycosylation changes in prostate cancer, highlighting its potential for biomarker discovery and improved understanding of cancer glyco-biology.