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 glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization 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 tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.
Papac
D I
DI
Department of Analytical Chemistry, Genentech, Inc., South San Francisco, CA 94080, USA.
Briggs
J B
JB
Chin
E T
ET
Jones
A J
AJ
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004563:Electrochemistry; D006023:Glycoproteins / Q000737:chemistry*; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification*; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D010959:Tissue Plasminogen Activator / Q000737:chemistry*
D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator