Characterisation of intact recombinant human erythropoietins applied in doping by means of planar gel electrophoretic techniques and matrix-assisted laser desorption/ionisation linear time-of-flight mass spectrometry

Authors

  • Gerald Stübiger,

    1. Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria
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  • Martina Marchetti,

    1. Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria
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  • Marietta Nagano,

    1. Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria
    2. ARC Seibersdorf Research, Doping Control Laboratory, A-2444 Seibersdorf, Austria
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  • Christian Reichel,

    1. ARC Seibersdorf Research, Doping Control Laboratory, A-2444 Seibersdorf, Austria
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  • Günter Gmeiner,

    1. ARC Seibersdorf Research, Doping Control Laboratory, A-2444 Seibersdorf, Austria
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  • Günter Allmaier

    Corresponding author
    1. Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria
    • Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A 1060 Vienna, Austria.
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  • Partly presented at 22nd Manfred Donike Workshop on Doping Analysis, Cologne (Germany), 7–12 March 2004, by G. Stübiger.

Abstract

Our experiments show that it is possible to detect different types of recombinant human erythropoietins (rhEPOs), EPO-α, EPO-β and novel erythropoesis stimulating protein (NESP), based on exact molecular weight (MW) determination by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) applying a high-resolution time-of-flight (TOF) mass analyser in the linear mode. Detection limits for the highly purified, intact glycoproteins were achievable in the low fmol range (25–50 fmol) using a sample preparation method applying a hydrophobic sample support (DropStop®) as MALDI target surface. These results are very promising for the development of highly sensitive detection methods for a direct identification of rhEPO after enrichment from human body fluids. During our investigation we were able to differentiate EPO-α, EPO-β and NESP based on distinct molecular substructures at the protein level by specific enzymatic reactions. MW determination of the intact molecules by high resolving one-dimensional sodium dodecyl sulfate /polyacrylamide gel electrophoresis (1D SDS-PAGE) and isoform separation by planar isoelectric focusing (IEF) was compared with MALDI-MS data. Migration differences between the rhEPOs were observed from gel electrophoresis, whereby MWs of 38 kDa in the case of EPO-α/β and 49 kDa for NESP could be estimated. In contrast, an exact MW determination by MALDI-MS based on internal calibration revealed average MWs of 29.8 ± 0.3 kDa for EPO-α/β and 36.8 ± 0.4 kDa for NESP. IEF separation of the intact rhEPOs revealed the presence of four to eight distinct isoforms in EPO-α and EPO-β, while four isoforms, which appeared in the more acidic area of the gels, were detected by immunostaining in NESP. A direct detection of the different N- or O-glycoform pattern from rhEPOs using MALDI-MS was possible by de-sialylation of the glycan structures and after de-N-glycosylation of the intact molecules. Thereby, the main glycoforms of EPO-α, EPO-β and NESP could be characterised based on their N-glycan composition. A microheterogeneity of the molecules based on the degree of sialylation of the O-glycan was observable directly from the de-N-glycosylated protein. Copyright © 2005 John Wiley & Sons, Ltd.

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