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J Sep Sci. 2005 Sep;28(14):1764-78.

Characterization of N- and O-glycopeptides of recombinant human erythropoietins as potential biomarkers for doping analysis by means of microscale sample purification combined with MALDI-TOF and quadrupole IT/RTOF mass spectrometry.

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1
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria.

Erratum in

  • J Sep Sci. 2005 Nov;28(16):2235.

Abstract

The structural characterization of the O- and N-glycan structures of three different commercially available recombinant human erythropoietins (rhEPOs) is represented by means of a microscale sample purification using ZipTip technology and MALDI-TOF and MALDI low-energy CID MS. Glycopeptides were released from rhEPO samples by a differential endoproteolytic digestion to obtain site-specific glycosylation patterns. Mass accuracies in the range of +/- 0.04% obtained by the high-resolution TOF instrument allowed an unambiguous assignment of N-glycan structures via glycan database software. Furthermore, the O-glycan structures were directly analyzed on the glycopeptide level by MS/MS experiments. Principally, site-specific glycosylation was found to be very similar for the three different rhEPOs (EPO-alpha, EPO-beta, and novel erythropoiesis stimulating protein (NESP)) but exhibiting quantitative differences in distinct O- and N-glycan moieties. Significant differences were found in the degree of sialylation and acetylation. Especially, a considerable degree of variation of the O-acetylation of sialic acid residues could be realized on the glycan structures of O- and N-glycopeptides, whereas EPO-alpha and EPO-beta could be clearly differentiated from NESP solely on the O-glycopeptide level.

PMID:
16224972
[Indexed for MEDLINE]

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