Optimization of Multiple Glycosidase and Chemical Stabilization Strategies for N-Glycan Isomer Detection by Mass Spectrometry Imaging in Formalin-Fixed, Paraffin-Embedded Tissues.

The analysis of N-glycan distributions in formalin-fixed, paraffin-embedded (FFPE) tissues by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an effective approach for characterization of many disease states. As the workflow has matured and new technology emerged, approaches are needed to more efficiently characterize the isomeric structures of these N-glycans to expand on the specificity of their localization within tissue. Sialic acid chemical derivatization can be used to determine the isomeric linkage (α2,3 or α2,6) of sialic acids attached to N-glycans, while endoglycosidase F3 (Endo F3) can be enzymatically applied to preferentially release α1,6-linked core fucosylated glycans, further describing the linkage of fucose on N-glycans. Here we describe workflows where N-glycans are chemically derivatized to reveal sialic acid isomeric linkages, combined with a dual-enzymatic approach of endoglycosidase F3 and PNGase F to further elucidate fucosylation isomers on the same tissue section.