Enzymatical hydrolysis of N-glycans from glycoproteins and fluorescent labeling by 2-aminobenzamide (2-AB).
When performing a structural analysis of N-glycans, a number of aspects should be considered. N-Glycans may be hydrolyzed from purifi ed glyc-oproteins, serum glycoprotein mixtures, or delipidated membrane fractions by chemical hydrolysis using hydrazine or enzymatic hydrolysis using PNGase F. Chemical deglycosylation may be an economical alternative to produce N-and O-glycans in a preparative scale, but it is less suitable for analytical purposes. By chemical hydrazinolysis the protein core is destroyed completely and all acyl groups are cleaved from neuraminic acid residues as well as from N-acetylhexosamine residues. If not only a structure analysis of N-glycans is intended but a sequencing of the protein core, an analysis of the different types of neuraminic acids or an elucidation of the carbohydrate structures in distinct glycosylation sites has to be performed in addition, enzymatical deglycosylation using PNGase F is the most suitable way to hydrolyze N-glycans from the protein backbone.
Nuck
Rolf
R
Institut für Molekularbiologie und Biochemie, Charité - Universitaetsmedizin Berlin, Berlin-Dahlem, Germany.
D005456:Fluorescent Dyes / Q000737:chemistry*; D006031:Glycosylation; D006868:Hydrolysis; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D062367:ortho-Aminobenzoates / Q000737:chemistry*
D005456:Fluorescent Dyes; D011134:Polysaccharides; D062367:ortho-Aminobenzoates; C000219:anthranilamide