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-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.

Evaluation of protein N‐glycosylation in 2‐DE: Erythropoietin as a study case

“…The experimental design proved reproducible both in terms of spot-number, spot-intensity, and relative position within the gel. As foreseen for the two molecules, rEPO-P migrated to a M r of ,35 kDa whereas NESP migrated to a M r of ,45 kDa, according to their hydrodynamic volume [18,28]. The influence of sialic acid residues to the separation in both dimensions is evident as it is the only factor contributing to the distinct pI values [29] between two adjacent IEF spots that also migrate to different M r values.…”

“…Spots were excised from the gel, minced, washed with water, shrunk in ACN for 10 min and dried in a vacuum centrifuge. Then, proteins contained in gel pieces were reduced and alkylated (necessary to com-pletely de-N-glycosylate NESP [18]), by adding 200 mL of 10 mM DTT in phosphate buffer (pH 7.3), left for 30 min at 567C, and the buffer replaced by a 50 mM IAA solution. After 30 min at room temperature (RT) in the dark with occasional agitation, gel pieces were washed with three cycles of dehydration with ACN, rehydration with phosphate and dried again in a vacuum centrifuge.…”

Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time‐of‐flight mass spectrometry

“…No significant differences in sialic acid content were found between the released glycans from epoetin-a and -b. However, intact epoetin-b presents two more basic sialoforms than epoetin-a ( [19,23]; Balaguer, E., Neusüß, C., Chromatographia 2006, submitted) and therefore, a higher content in less sialylated glycans was expected. This disagreement can be explained by the fact that sialylation differences between both epoetins can be situated in the O-glycans that have not been analysed here.…”

“…Structural characterization of rHuEPO has been usually performed by peptide mapping after enzymatic digestion [2,[8][9][10] or glycan analysis after chemical or enzymatic release [11][12][13]. At the intact protein level, rHuEPO has been mainly analysed by CZE [14][15][16][17], IEF [18][19][20], 2-DE [21], CIEF [22] and MALDI-TOF-MS [23]. Nevertheless, the structural information provided by these methods is limited.…”

Assessing the instability of the isoelectric focusing patterns of erythropoietin in urine

“…Mass spectrometric experiments after deglycosylation were performed as described before [19] using a Voyager-DE ™ STR Biospectrometry workstation (Applied Biosystems).…”