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

✍ Scribed by Gerald Stübiger; Martina Marchetti; Marietta Nagano; Rudolf Grimm; Günter Gmeiner; Christian Reichel; Günter Allmaier

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 411 KB
• Volume: 28
• Category: Article
• ISSN: 1615-9306
• DOI: 10.1002/jssc.200500148

✦ Synopsis

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‐α, EPO‐β, 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‐α and EPO‐β could be clearly differentiated from NESP solely on the O‐glycopeptide level.