Our experiments show that it is possible to detect different types of recombinant human erythropoietins (rhEPOs), EPO-a, EPO-b 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 1 ) 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-a, EPO-b 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-a/b 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 AE 0.3 kDa for EPO-a/b and 36.8 AE 0.4 kDa for NESP. IEF separation of the intact rhEPOs revealed the presence of four to eight distinct isoforms in EPO-a and EPO-b, 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-a, EPO-b 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.

See original 2005, vol. 28, issue 14, pp. 1764–1778. DOI 10.1002/jssc.200500148

A screening method to determine the binding behavior of lectins toward recombinant human erythropoietin (rHuEPO) was developed. Twenty-three different lectins were tested for this purpose. rHuEPO isoforms were separated by isoelectric focusing using the International Olympic Committee (IOC) and World Anti-Doping Agency (WADA) accredited method for the direct detection of the prohibited doping substance erythropoietin (EPO). For the visualization of the rHuEPO isoforms lectins were used instead of antibodies. Optimization of the screening protocol enabled the detection of a maximum number of rHuEPO isoforms. By means of this protocol information about the binding properties of a lectin toward each individual rHuEPO isoform was accessible. All evaluated lectins showed significant differences in their binding behavior. The most intense response was obtained with WGA, DSL, PHA-E, LEL, PSA, and LCA. While WGA, DSL, PHA-E, and LEL were able to bind all isoforms detected by the standard antibody, LCA and PSA demonstrated a clear preference for rHuEPO isoforms located in the more basic region of the electropherogram. Further lectins tested were ConA, succWGA, PHA-L, RCA, SNA, MAA, STL, ECL, GSL-II, SJA, SBA, UEA-I, Jacalin, PNA, DBA, GSL-I, and VVA. Compared to the lectins mentioned above, they showed reduced sensitivity. Endogenous and recombinant EPO only differ in the composition of their N- and O-glycan moieties. As lectins possess the unique ability to recognize subtle differences in glycan substructures, they represent an interesting approach for their structural characterization. Furthermore, they might be useful for affinity enrichment/purification of rHuEPO in doping control.

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.