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

J Sep Sci. 2005 Sep;28(14):1764-78. doi: 10.1002/jssc.200500148.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Erythropoietin / chemistry*
  • Erythropoietin / isolation & purification
  • Freeze Drying / methods
  • Glycopeptides / chemistry*
  • Glycopeptides / isolation & purification
  • Humans
  • Indicators and Reagents
  • Mass Spectrometry / methods
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Recombinant Proteins
  • Solvents
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods


  • Glycopeptides
  • Indicators and Reagents
  • Peptide Fragments
  • Recombinant Proteins
  • Solvents
  • Erythropoietin