Detecting Mannosidase Activities Using Ribonuclease B and Matrix-Assisted Laser Desorption/Ionization–Time of Flight Mass Spectrometry
| Autor: | David Beighton, Karen A. Homer, Michael Wilson, Gretta Roberts, H. L. Byers, Edward Tarelli |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
PNGase F
Mannosidase RNase P Population Biophysics Mass spectrometry Biochemistry Amidohydrolases Ribonucleases Mannosidases Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Ribonuclease education Molecular Biology education.field_of_study Chromatography Bacteria biology Chemistry Cell Biology Culture Media Hexosaminidases Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Time-of-flight mass spectrometry Mannose |
| Zdroj: | Analytical Biochemistry. 282:165-172 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.2000.4606 |
| Popis: | 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 GlcNAcbond;Asn and GlcNAcbond;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. |
| Databáze: | OpenAIRE |
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