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

ABSTRACT: Here we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of MALDI N-glycan 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, making our study the first report that directly links 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 slides 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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Canis Familiaris (dog) (canis Lupus Familiaris)

TISSUE(S): Brain

DISEASE(S): Brain Cancer

SUBMITTER: Stacy Malaker

LAB HEAD: Stacy Malaker

PROVIDER: PXD025537 | Pride | 2022-05-04

REPOSITORIES: Pride

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			Action	DRS
	110218_SAM_VH13_0935_benign.raw	Raw
	110218_SAM_VH13_0935_benign.raw_20190103_Oglycan.xlsx	Xlsx
	110218_SAM_VH13_0935_benign.raw_20190103_Omannose.xlsx	Xlsx
	110218_SAM_VH13_0935_tumor.raw	Raw
	110218_SAM_VH13_0935_tumor.raw_20190103_Omannose.xlsx	Xlsx

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

Project description:Here we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of MALDI N-glycan 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, making our study the first report that directly links 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 slides 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.

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