Low LC-MS/MS detection of glycopeptides released from pmol levels of recombinant erythropoietin using nanoflow HPLC-chip electrospray ionization.
The test used by anti-doping laboratories to detect the misuse of recombinant erythropoietin (rhEPO) is based on its different migration pattern on isoelectric focusing (IEF) gel compared with the endogenous human erythropoietin (hEPO) that can possibly be explained by structural differences. While there is definitely a need to identify those differences by LC-MS/MS, the extensive characterization that was achieved for the rhEPO was never performed on human endogenous EPO because its standard is not available in sufficient amount. The goal of this study was to develop an analytical method to detect pmol amounts of N-linked and O-linked glycopeptides of the recombinant hormone as a model. Using a nanoflow HPLC-Chip electrospray ionization/ion trap mass spectrometer, the diagnostic ion at m/z 366 of oligosaccharides was monitored in the product ion spectra to identify the four theoretical glycosylation sites, Asn24, Asn38, Asn83 and Ser126, respectively, on glycopeptides 22-37, 38-55, 73-96 and 118-136. With 3 pmol of starting material applied on Chip, only the desialylated N-glycopeptides 22-37 and 38-55/38-43 could be observed, and of all the glycan isoforms, those with the smaller structures were predominantly detected. While the preservation of the sialic acid moieties decreased the detection of all the N-glycopeptides, it allowed a more extensive characterization of the O-linked glycopeptide 118-136. The technique described herein provides a mean to detect glycopeptides from commercially available pharmaceutical preparations of rhEPO with the sensitivity required to analyze pmol amounts of hEPO, which could ultimately lead to the identification of structural differences between the recombinant and the human forms of the hormone.
Laboratoire de contrôle du dopage, INRS-Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, Québec, Canada. pauleemilie.groleau@iaf.inrs.ca
Paule Emilie
PE
Groleau
Philippe
P
Desharnais
Linda
L
Coté
Christiane
C
Ayotte
D002851:Chromatography, High Pressure Liquid; D004300:Doping in Sports; D004921:Erythropoietin / Q000652:urine*; D006020:Glycopeptides / Q000652:urine*; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods; D011237:Predictive Value of Tests; D011994:Recombinant Proteins; D021241:Spectrometry, Mass, Electrospray Ionization; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry
D006020:Glycopeptides; D011994:Recombinant Proteins; D004921:Erythropoietin