title: 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.
abstract: Our experiments show that it is possible to detect different types of recombinant human erythropoietins (rhEPOs), EPO-alpha, EPO-beta 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-alpha, EPO-beta 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-alpha/beta 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-alpha/beta 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-alpha and EPO-beta, 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-alpha, EPO-beta 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.
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria.
authors.forename: Gerald
authors.initials: G
authors.lastname: Stübiger
authors.forename: Martina
authors.initials: M
authors.lastname: Marchetti
authors.forename: Marietta
authors.initials: M
authors.lastname: Nagano
authors.forename: Christian
authors.initials: C
authors.lastname: Reichel
authors.forename: Günter
authors.initials: G
authors.lastname: Gmeiner
authors.forename: Günter
authors.initials: G
authors.lastname: Allmaier
mesh_terms: D004300:Doping in Sports / Q000517:prevention & control*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004921:Erythropoietin / Q000032:analysis* / Q000737:chemistry*; D006801:Humans; D011994:Recombinant Proteins; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D015813:Substance Abuse Detection / Q000379:methods*
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin