Ribonuclease
(RNase) B incubated with purified enzymes, whole bacterial cultures, or their separated components-cells and supernates-have been directly analyzed by matrix-assisted laser desorption/ ionization-time of flight mass 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 ␣-(1 3 2)-mannosidase activity converts the glycoprotein to its Man 5 form, identifiable by its mass of 14,899 [M ؉ H] ؉ ; this species subsequently is converted, by the actions of ␣-(1 3 3) and ␣-(1 3 6)-mannosidases, to the Man 1 form via Man 4 , Man 3 , and Man 2 . The Man 1 glycoform (which is readily isolated) has then similarly been used for identifying -(1 3 4)-mannosidase and the derived Man 0 form has served in turn as a natural substrate for -(1 3 4) N-acetylglucosaminidase producing a species possessing a single asparagine-linked GlcNAc residue (mass 13,886). Mannose liberated from the actions of mannosidases can, if desired, be quantified by, for example, chromatography. The actions and specificities of endoglycosidases such as a peptide-N-glycosidase F (PNGase F) and of endo-N-acetlyglucosaminidases (e.g., endo-F and endo-H), which respectively cleave between the GlcNAcOAsn and GlcNAcOGlcNAc bonds of N-linked glycoproteins, are also demonstrable by MALDI-ToF analysis of RNase B (and derived products). From these digests the completely deglycosylated polypeptide corresponding to RNase A in which Asn has been converted to Asp (mass 13,684) and a species corresponding to RNase A ؉ GlcNAc (mass 13,886) are produced, together with their correspond-ing free oligosaccharides which are amenable to analysis by both MALDI-ToF and by HPLC.