On-slide tissue digestion for mass spectrometry based glycomic and proteomic profiling

Abstract We describe a protocol for glycomic and proteomic profiling that uses serial enzyme digestions from the surface of fresh frozen or fixed tissue slides. The abundances of the extracted glycans and peptides are determined using liquid chromatography-mass spectrometry. In a typical experiment, our method quantifies 14 heparan sulfate disaccharides, 11 chondroitin sulfate disaccharides, 50 N-glycan compositions and approximately 1200 proteins from a 1.8 mm circle, on fresh frozen rat brain. Each enzymatic digestion is incubated overnight with direct application of enzyme on the tissue surface. Overall, the sample preparation process for multiple tissue slides takes a day per biomolecule class. This protocol saves time by simultaneous digestion of large N-glycans and small HS disaccharides and subsequent separation using size exclusion chromatography. Compared to wet tissue analysis, this method requires less time by a factor of two. By comparison, MALDI-imaging provides higher spatial resolution of glycans and proteins but lower depth of coverage. MALDI dissociates fragile glycan substituents including sulfates and is not recommended for analysis of glycosaminoglycans (GAGs).