Detecting mannosidase activities using ribonuclease B and matrix-assisted laser desorption/ionization-time of flight mass spectrometry

Ribonuclease (RNase) B incubated with purified enzymes, whole bacterial cul tures, or their separated components-cells and supernates-have been directl y analyzed by matrix-assisted laser desorption/ionization-time of flight ma ss spectrometry (MALDI-ToF) to detect exomannosidases and to evaluate their specificities and location. Enzymatic cleavage was monitored by observing changes in RNase B glycoform population. Thus a nonspecific alpha-(1 --> 2) -mannosidase activity converts the glycoprotein to its Man(5) form, identif iable by its mass of 14,899 [M + H](+); this species subsequently is conver ted, by the actions of alpha-(1 --> 3) and alpha-(1 --> 6)-mannosidases, to the Man(1) form via Man(4), Man(3), and Man(2). The Man(1) glycoform (whic h is readily isolated) has then similarly been used for identifying beta-(1 --> 4)-mannosidase and the derived Man(0) form has served in turn as a nat ural substrate for beta-(1 --> 4) N-acetylglucosaminidase producing a speci es possessing a single asparagine-linked GlcNAc residue (mass 13,886). Mann ose liberated from the actions of mannosidases can, if desired, be quantifi ed by, for example, chromatography. The actions and specificities of endogl ycosidases such as a peptide-N-glycosidase F (PNGase F) and of endo-N-acetl yglucosaminidases (e.g., endo-F and endo-H), which respectively cleave betw een the GlcNAc-Asn and GlcNAc-GlcNAc bonds of N-linked glycoproteins, are a lso demonstrable by MALDI-ToF analysis of RNase B land derived products). F rom these digests the completely deglycosylated polypeptide corresponding t o RNase A in which Asn has been converted to Asp (mass 13,684) and a specie s corresponding to RNase A + GlcNAc (mass 13,886) are produced, together wi th their corresponding free oligosaccharides which are amenable to analysis by both MALDI-ToF and by HPLC. (C) 2000 Academic Press.