title: Detecting mannosidase activities using ribonuclease B and matrix-assisted laser desorption/ionization-time of flight mass spectrometry.
abstract: 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 alpha-(1 --> 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 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 (which 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 natural substrate for beta-(1 --> 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 GlcNAc&bond;Asn and GlcNAc&bond;GlcNAc 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 corresponding free oligosaccharides which are amenable to analysis by both MALDI-ToF and by HPLC.
authors.affiliation: Joint Microbiology Research Unit, Guy's, King's & St Thomas's Dental Institute, Caldecot Road, Denmark Hill, London, SE5 9RW, United Kingdom. ted.tarelli@kcl.ac.uk
authors.forename: E
authors.initials: E
authors.lastname: Tarelli
authors.forename: H L
authors.initials: HL
authors.lastname: Byers
authors.forename: M
authors.initials: M
authors.lastname: Wilson
authors.forename: G
authors.initials: G
authors.lastname: Roberts
authors.forename: K A
authors.initials: KA
authors.lastname: Homer
authors.forename: D
authors.initials: D
authors.lastname: Beighton
mesh_terms: D000581:Amidohydrolases / Q000032:analysis*; D001419:Bacteria / Q000201:enzymology; D003470:Culture Media; D006596:Hexosaminidases / Q000032:analysis*; D008358:Mannose / Q000032:analysis; D008361:Mannosidases / Q000032:analysis*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012260:Ribonucleases / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D003470:Culture Media; D012260:Ribonucleases; C029600:ribonuclease B; D006596:Hexosaminidases; D008361:Mannosidases; C412205:beta-1,4-mannosidase; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008358:Mannose