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

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.