On-tissue amidation of sialic acid with aniline for sensitive imaging of sialylated N-glycans from FFPE tissue sections via MALDI mass spectrometry

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Abstract
Spatial visualization of glycans within clinical tissue samples is critical for discovery of disease-relevant glycan dysregulations. Herein, we develop an on-tissue derivatization strategy for sensitive spatial visualization of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections, based on amidation of sialic acid residues with aniline. The sialylated N-glycans were stabilized and given enhanced signal intensity owing to selective capping of a phenyl group to the sialic acid residue after aniline labeling. Proof-of-concept experiments, including determinations of sialylglycopeptide and N-glycans enzymatically released from glycoproteins, were performed. Further, mass spectrometry (MS) imaging of N-glycans on human laryngeal cancer FFPE tissue sections was conducted via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), based on our strategy for on-tissue amidation of sialylated N-glycans. We obtained higher sialylated N-glycan coverages for both the glycoproteins and cancer tissue samples, demonstrating that the detection sensitivity for sialylated N-glycans is notably improved by amidation derivatization. We also characterized N-glycan heterogeneity across the human laryngeal cancer tissue section, showing N-glycan dysregulation in the tumor region. Graphical abstract

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