Low LC‐MS/MS detection of glycopeptides released from pmol levels of recombinant erythropoietin using nanoflow HPLC‐chip electrospray ionization

Abstract

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.

Detection of EPO doping and blood doping: the haematological module of the Athlete Biological Passport

“…[17][18][19] Up to now, both approaches give very promising results in laboratory conditions, but have not yet been fully validated, accredited, and accepted by WADA. [17][18][19] Up to now, both approaches give very promising results in laboratory conditions, but have not yet been fully validated, accredited, and accepted by WADA.…”

The Analytical Chemistry of Drug Monitoring in Athletes

“…Because the carbohydrate portion of the EPO molecule distinguishes native EPO from pharmaceutical EPO, there have been attempts to characterize the glycosylation of EPO after separation by various techniques (146)(147)(148). MALDI-MS combined with a high-resolution TOF mass analyzer was used to detect purified intact EPO in the low femtomolar range (25-50 fmol) (149).…”

Miniaturized Systems for Analytical Separations I: Systems Based on a Hydrodynamic Flow

“…Groleau and coworkers developed an analytical method to detect pmol amounts of N-linked and O-linked glycopeptides of the recombinant hormone (used as a model), using a nanoflow HPLC Chip-ESI-ITMS [52]. 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 hEPO because its standard is not available in sufficient amount.…”