A HIGH-THROUGHPUT MICROSCALE METHOD TO RELEASE, N-LINKED OLIGOSACCHARIDES FROM GLYCOPROTEINS FOR MATRIX-ASSISTED-LASER-DESORPTION IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRIC ANALYSIS/

Citation
Di. Papac et al., A HIGH-THROUGHPUT MICROSCALE METHOD TO RELEASE, N-LINKED OLIGOSACCHARIDES FROM GLYCOPROTEINS FOR MATRIX-ASSISTED-LASER-DESORPTION IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRIC ANALYSIS/, Glycobiology, 8(5), 1998, pp. 445-454
Citations number
21
Categorie Soggetti
Biology
Journal title
ISSN journal
09596658
Volume
8
Issue
5
Year of publication
1998
Pages
445 - 454
Database
ISI
SICI code
0959-6658(1998)8:5<445:AHMMTR>2.0.ZU;2-B
Abstract
This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of gly coproteins, A 96-well MultiScreen assay system containing a polyvinyli dene difluoride (PVDF) membrane is employed to immobilize glycoprotein s for subsequent enzymatic deglycosylation. Recombinant tissue-type pl asminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 mu g of a glycoprotein, This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatical ly with peptide N-glycosidase F (PNGaseF) from as little as 0.5 mu g r t-PA for subsequent analysis by matrix-assisted laser desorption/ioniz ation time-of-flight (MALDI-TOF) mass spectrometry, The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF- mediated release of oligosaccharides from rt-PA as determined by trypt ic mapping experiments. Comparison of the oligosaccharides released fr om 50 mu g of rt-PA by either the 96-well plate method or by a standar d solution digestion procedure showed no significant differences in th e profiles obtained by high-pH anion-exchange chromatography with puls ed amperometric detection (HPAEC-PAD), Both neutral and sialylated oli gosaccharide standards spiked into wells were recovered equally as det ermined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoprote ins with easy removal of reagents prior to PNGaseF digestion. In addit ion, this method allows 60 glycoprotein samples tell be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.