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 understanding dysregulated glycosylation. [Display omitted] • N-glycan MALDI-MSI revealed a sialylated glycan as upregulated in canine glioma • Spatial glycoproteomics identified haptoglobin as the underlying glycoprotein • Manual assignment of glycopeptides uncovered a diverse array of glycosylation • Observed significant glycopeptide changes between tumor and benign regions Malaker et al. use a multi-omics approach, including MALDI-MSI and glycoproteomics, to understand the spatial distribution of glycans and their associated glycoproteins in canine glioma. [ABSTRACT FROM AUTHOR]