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.
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.
Stübiger
Gerald
G
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria.
Marchetti
Martina
M
Nagano
Marietta
M
Grimm
Rudolf
R
Gmeiner
Günter
G
Reichel
Christian
C
Allmaier
Günter
G
D004921:Erythropoietin / Q000737:chemistry* / Q000302:isolation & purification; D005612:Freeze Drying / Q000379:methods; D006020:Glycopeptides / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins; D012997:Solvents; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D006020:Glycopeptides; D007202:Indicators and Reagents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012997:Solvents; D004921:Erythropoietin