On-tissue spatially resolved glycoproteomics guided by N-glycan imaging reveal global dysregulation of canine glioma glycoproteomic landscape

Here, we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of matrix-assisted laser desorption ionization (MALDI) N-glycan mass spectrometry imaging (MSI) and spatially resolved glycoproteomics. We subjected glioma biopsies to on-tissue PNGaseF digestion and MALDI-MSI and found that the glycan HexNAc4-Hex5-NeuAc2 was predominantly expressed in necrotic regions of high-grade canine gliomas. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were subjected to microdigestion and manual glycoproteomic analysis. Results identified haptoglobin as the protein associated with HexNAc4-Hex5-NeuAc2, thus directly linking glycan imaging with intact glycopeptide identification. In total, our spatially resolved glycoproteomics technique identified over 400 N-, O-, and S-glycopeptides from over 30 proteins, demonstrating the diverse array of glycosylation present on the tissue slices and the sensitivity of our technique. Ultimately, this proof-of -principle work demonstrates that spatially resolved glycoproteomics greatly complement MALDI-MSI in under

Automated summary

In this study, we propose an approach combining MALDI N-glycan mass spectrometry imaging (MSI) with spatially resolved glycoproteomics to identify N-linked glycoproteins and deduce their spatial localization. The experiments conducted on glioma biopsies revealed the predominant expression of a specific glycan in necrotic regions of high-grade canine gliomas, which was further linked to a specific protein through manual glycoproteomic analysis. Our spatially resolved glycoproteomics technique successfully identified over 400 glycopeptides from various proteins, demonstrating the diversity of glycosylation on tissue slices and the sensitivity of the technique.