MALDI imaging mass spectrometry mapping of the glycocalyx.

The glycocalyx is a dense complex matrix of glycoproteins, glycosaminoglycans and glycolipids on the cell surface involved in maintaining cell integrity and interactions with the cellular microenvironment. Alterations to the glycan constituents of the glycocalyx are implicit in progression of diseases like cancer, but its overall complexity makes biomolecular analysis very challenging. Changes in cell surface protein glycosylation are common alterations that occur with tumor progression and reflect the clinical diagnostic detection of many cancer biomarkers like prostate specific antigen and CA19-9. There is also an underlying metabolic connection to cancer glycosylation changes and the well described changes in tumor metabolism linked with glucose and glutamine utilization. Structurally, these potential tumor glycans are represented by multiple sialylated and fucosylated species including known tumor antigens such as sialyl Lewis X, sialyl Lewis A and Lewis Y motifs. Imaging mass spectrometry of clinical tissues and new enzymatic and chemical sample preparation strategies have provided new approaches for spatial characterization of the glycocalyx constituents. N-glycan MALDI imaging MS allows direct histopathology localization of these glycans to tumor, stroma and immune regions. Peptide N-glycosidase and other glycosidases are sprayed directly on tissues, and released glycans are detected spatially on a MALDI mass spectrometer. The method was developed to work on clinical formalin-fixed paraffin embedded tissue slides readily obtainable from pathology services. Generally, 45-60 N-glycan species are detected in most tissues, and these numbers can increase to 100 or more in advanced cancer tissues. Example data from prostate, colon, and pancreatic cancer tissues will be presented, including new workflows that specifically target identification of isomeric species of branched fucosylated and sialylated N-glycans. This includes combined use of immunohistochemistry staining of known carbohydrate tumor antigens prior to N-glycan imaging, performed on the same slide. Strategies to use the tissue N-glycan distribution maps for glycoproteomic and cancer biomarker analysis will also be presented. Additional glycosidases, other enzymes and other imaging modalities can also be incorporated into the established workflows to better define the complex organization of the tissue glycocalyx.