Specific alterations in N-linked glycans, such as core fucosylation, are associated with many cancers and other disease states. Because of the many possible anomeric linkages associated with fucosylated -glycans, determination of specific anomeric linkages and the site of fucosylation (i.e., core vs outer arm) can be difficult to elucidate. A new MALDI mass spectrometry imaging workflow in formalin-fixed clinical tissues is described using recombinant endoglycosidase F3 (Endo F3), an enzyme with a specific preference for cleaving core-fucosylated -glycans attached to glycoproteins. In contrast to the broader substrate enzyme peptide--glycosidase F (PNGaseF), Endo F3 cleaves between the two core -acetylglucosami... More
Specific alterations in N-linked glycans, such as core fucosylation, are associated with many cancers and other disease states. Because of the many possible anomeric linkages associated with fucosylated -glycans, determination of specific anomeric linkages and the site of fucosylation (i.e., core vs outer arm) can be difficult to elucidate. A new MALDI mass spectrometry imaging workflow in formalin-fixed clinical tissues is described using recombinant endoglycosidase F3 (Endo F3), an enzyme with a specific preference for cleaving core-fucosylated -glycans attached to glycoproteins. In contrast to the broader substrate enzyme peptide--glycosidase F (PNGaseF), Endo F3 cleaves between the two core -acetylglucosamine residues at the protein attachment site. On tissues, this results in a mass shift of 349.137 a.m.u. for core-fucosylated -glycans when compared to -glycans released with standard PNGaseF. Endo F3 can be used singly and in combination with PNGaseF digestion of the same tissue sections. Initial results in liver and prostate tissues indicate core-fucosylated glycans associated to specific tissue regions while still demonstrating a diverse mix of core- and outer arm-fucosylated glycans throughout all regions of tissue. By determining these specific linkages while preserving localization, more targeted diagnostic biomarkers for disease states are possible without the need for microdissection or solubilization of the tissue.
