In Situ Imaging of O-Linked β-N-Acetylglucosamine Using On-Tissue Hydrolysis and MALDI Mass Spectrometry

SIMPLE SUMMARY: Post-translational O-glycosylation of proteins with N-acetylglucosamine serves as a cellular regulator that has been linked to cancer. This sugar’s variability is emerging as a metabolic biomarker in cancer, and here we investigate the use of mass spectrometry imaging to visualize the location of this sugar in primary tumor sections. O-GlcNAc hydrolase, an enzyme specific to N-acetylglucosamine, has been employed for on-tissue hydrolysis of this sugar, providing additional evidence implicating it in tumor growth. ABSTRACT: Post-translational O-glycosylation of proteins via the addition of N-acetylglucosamine (O-GlcNAc) is a regulator of many aspects of cellular physiology. Processes driven by perturbed dynamics of O-GlcNAcylation modification have been implicated in cancer development. Variability in O-GlcNAcylation is emerging as a metabolic biomarker of many cancers. Here, we evaluate the use of MALDI-mass spectrometry imaging (MSI) to visualize the location of O-GlcNAcylated proteins in tissue sections by mapping GlcNAc that has been released by the enzymatic hydrolysis of glycoproteins using an O-GlcNAc hydrolase. We use this strategy to monitor O-GlcNAc within hepatic VX2 tumor tissue. We show that increased O-GlcNAc is found within both viable tumor and tumor margin regions, implicating GlcNAc in tumor progression.