Glycosite Mapping and in situ Mass Spectrometry Imaging of MUC2 Glycopeptides via On-slide Digestion with Mucinase StcE

Many cancers are characterized by altered mucin expression and glycosylation, although the mechanistic relationship between tumor glycosylation and disease progression is not well-defined. Herein, our goal was to map specific mucin glycoforms in diseased tissue, enabling correlation of the tumor glycan profile with malignant features. To this end, we developed a workflow implementing on-tissue digestion with mucinase StcE, followed by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and liquid chromatography coupled to mass spectrometry (LC-MS). To optimize our workflow, we analyzed four different mucinous carcinomas derived from colon, esophageal, and salivary gland tissue. Using this technique, we deduced the spatial distribution of StcE-generated O-glycopeptides within mucinous tumors using MALDI-IMS. Subsequent LC-MS analyses revealed the identity of different species detected in imaging experiments, in addition to comprehensively characterizing the mucinome and proteome of each tissue. Our coupled MS approach unveiled a striking mucin 2 (MUC2) expression pattern in two colorectal mucinous adenocarcinomas, in which different glycoforms clearly stratified regions within the tumor. Notably, our LC-MS experiments obtained near-complete sequence coverage over the mucin domains of MUC2, enabling glycoproteomic mapping of this canonical mucin in unprecedented depth. MUC2 glycosylation was dominated by the T and Tn antigens, with surprisingly little sialylation detected. However, O-glycans containing mono- and di-O-acetylated sialic acid were detected in low abundance. Finally, we obtained spectral evidence for an endogenous O-acetylated GalNAc, an O-glycan structure not previously reported in the literature. Overall, this proof-of-concept work underscores the potential of this technique to generate new research avenues in oncology and beyond. Significance StatementAberrant mucin expression and glycosylation are hallmarks of cancer, but how these changes promote malignant processes are not well understood. Solid tumors are highly heterogeneous in their cellular and molecular composition, and many advanced spatial techniques have emerged in recent years to study the tumor microenvironment (TME) for better understanding disease progression. Spatially resolved glycoprotein analyses typically detect either the protein or glycan components, but not both. We developed a workflow using a dual mass spectrometry approach to map the location of intact glycopeptides in mucinous tumors, enabled by on-tissue digestion with the mucin-specific protease StcE. Future applications of this method on larger patient cohorts will enhance our understanding of glycans in malignancy, identify disease biomarkers, and define therapeutic targets.