On-line high-performance liquid chromatography/mass spectrometric characterization of native oligosaccharides from glycoproteins |
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Authors: | Barroso Begona Dijkstra Rene Geerts Marlieke Lagerwerf Fija van Veelen Peter de Ru Arnoud |
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Institution: | Pharming Group N.V., Archimedesweg 4, 2333 CN Leiden, The Netherlands. b.barroso@farm.rug.nl |
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Abstract: | 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. |
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