A monolithic PNGase F enzyme microreactor enabling glycan mass mapping of glycoproteins by mass spectrometry.
We demonstrate a simple and rapid single-step method to fabricate an enzyme microreactor incorporating the N-glycosidase PNGase F (peptide-N-glycosidase F) into a porous polymer-based monolith. The monolith is contained in a capillary format, while the enzyme reactor is ready to use within 1 h of preparation. The monomers making up the monolith, including N-acryloxysuccinimide for covalent immobilization of the enzyme, are mixed with PNGase F and introduced into the column by capillary force for polymerization/immobilization. Glycoproteins (ribonuclease B, asialofetuin, alpha1-acid glycoprotein, and ovalbumin) perfused through the PNGase F reactor were shown to be effectively deglycosylated on a time-scale of seconds/low minutes using low nanogram to microgram per microliter concentration (corresponding to a total sample consumption of 0.1-20 microg of a glycoprotein). The reactor enzyme activity was shown to be reproducible for at least 8 weeks when used and stored at room temperature. Evaluation was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Palm
Anders K
AK
Department of Chemistry, Indiana University, Bloomington, IN 47405, USA. novotny@indiana.edu
Novotny
Milos V
MV
D001212:Asialoglycoproteins / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry; D060748:Fetuins; D006023:Glycoproteins / Q000737:chemistry*; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods; D009961:Orosomucoid / Q000737:chemistry; D010047:Ovalbumin / Q000737:chemistry; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis*; D015203:Reproducibility of Results; D012260:Ribonucleases / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D000509:alpha-Fetoproteins / Q000737:chemistry
D001212:Asialoglycoproteins; D004800:Enzymes, Immobilized; D060748:Fetuins; D006023:Glycoproteins; D009961:Orosomucoid; D011134:Polysaccharides; D000509:alpha-Fetoproteins; C017029:asialofetuin; D010047:Ovalbumin; D012260:Ribonucleases; C029600:ribonuclease B; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase