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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
Author(s) -
Stübiger Gerald,
Marchetti Martina,
Nagano Marietta,
Grimm Rudolf,
Gmeiner Günter,
Reichel Christian,
Allmaier Günter
Publication year - 2005
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200500148
Subject(s) - glycan , glycopeptide , chemistry , glycosylation , peptide , acetylation , sialic acid , glycomics , mass spectrometry , recombinant dna , glycoprotein , biochemistry , chromatography , gene , antibiotics
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.