On-line high-performance liquid chromatography/mass spectrometric characterization of native oligosaccharides from glycoproteins.
An on-line high-performance liquid chromatography/mass spectrometry (HPLC/MS) method is described for the rapid characterization of any type of oligosaccharide released from glycoproteins. The procedure can be applied without further manipulation to fractions collected from a high-performance anion-exchange chromatography-pulse amperometric detection (HPAEC-PAD) system commonly used for glycosylation mapping of glycoproteins, or to a pool of oligosaccharides directly released from glycoproteins. The system consists of a porous graphitized high-performance chromatography column (Hypercarb) coupled to a quadrupole time-of-flight (TOF) mass spectrometer. Oligosaccharides are eluted from the column with a gradient of ammonium acetate/acetonitrile and directly identified following in-source fragmentation. Some applications of the method are presented, as well as information about the spectra and fragmentation behavior observed for N- and O-linked oligosaccharides released from some recombinant glycoproteins. Low femtomole limits of detection are achieved using proper miniaturization.
Barroso
Begona
B
Pharming Group N.V., Archimedesweg 4, 2333 CN Leiden, The Netherlands. b.barroso@farm.rug.nl
Dijkstra
Rene
R
Geerts
Marlieke
M
Lagerwerf
Fija
F
van Veelen
Peter
P
de Ru
Arnoud
A
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009844:Oligosaccharides / Q000032:analysis*
D006023:Glycoproteins; D009844:Oligosaccharides