Complete analysis of the glycosylation and disulfide bond pattern of humanbeta-hexosaminidase B by MALDI-MS

beta -hexosaminidase B is an enzyme that is involved in the degradation of glycolipids and glycans in the lysosome, Mutation in the HEXB gene lead to Sandhoff disease, a glycolipid storage disorder characterized by severe neu rodegeneration. So far, little structural information on the protein is ava ilable. Here, the complete analysis of the disulfide bond pattern of the pr otein is described for the first time, Additionally, the structures of the N-glycans are analyzed for the native human protein and for recombinant pro tein expressed in SF21 cells, For the analysis of the disulfide bond structure, the protein was proteolyt ically digested and the resulting peptides were analyzed by MALDI-MS. The a nalysis revealed three disulfide bonds (C91-C137; C309-C360; C534-C551) and a free cysteine (C487), The analysis of the N-glycosylation was performed by tryptic digestion of the protein, isolation of glycopeptides by lectin c hromatography and mass measurement before and after enzymatic deglycosylati on, Carbohydrate structures were calculated from the mass difference betwee n glycosylated and deglycosylated peptide. For beta -hexosaminidase B from human placenta, four N-glycans were identified and analyzed, whereas the re combinant protein expressed in SF21 cells carried only three glycans, In bo th cases the glycosylation belongs to the mannose-core- or high-mannose-typ e, and some carbohydrate structures are fucosylated.