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