title: Desialylation improves the detection of recombinant erythropoietins in urine samples analyzed by SDS-PAGE.
abstract: Recombinant erythropoietin (rhEPO) has been misused for over two decades by athletes, mainly but not only in endurance sports. A direct rhEPO detection method in urine by isoelectric focusing (IEF) was introduced in 2000, but the emergence of third-generation erythropoiesis-stimulating agents and so-called biosimilar rhEPOs, together with the sensitivity of human endogenous EPO (huEPO) pattern to enzymatic activities and its modification following short strenuous exercise, prompted the development of a complementary test based on SDS-PAGE analysis. While Mircera and NESP are easily detected with the existing IEF and SDS-PAGE methods, some samples containing both epoetin-α/β and huEPO present profiles that are still difficult to interpret. As doping practices have moved to micro-dosing, these mixed patterns are more frequently observed. We investigated the impact of enzymatic desialylation on the urinary and serum EPO profiles obtained by SDS-PAGE with the aim of improving the separation of the bands in these mixed EPO populations. We observed that the removal with neuraminidase of the sialic acid moieties from the different EPOs studied reduced their apparent molecular weight (MW) and increased the migration distance between huEPO and rhEPO centroids, therefore eliminating the size overlaps between them and improving the detection of rhEPO.
authors.affiliation: Laboratoire de contrôle du dopage, INRS-Institut Armand-Frappier, Laval, Québec, Canada.
authors.forename: Philippe
authors.initials: P
authors.lastname: Desharnais
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Naud
authors.forename: Christiane
authors.initials: C
authors.lastname: Ayotte
mesh_terms: D003016:Clostridium perfringens / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004921:Erythropoietin / Q000097:blood* / Q000737:chemistry / Q000378:metabolism / Q000652:urine*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D019158:N-Acetylneuraminic Acid / Q000302:isolation & purification* / Q000378:metabolism; D009439:Neuraminidase / Q000378:metabolism; D020033:Protein Isoforms / Q000097:blood / Q000737:chemistry / Q000378:metabolism / Q000652:urine; D011994:Recombinant Proteins / Q000097:blood / Q000737:chemistry / Q000378:metabolism / Q000652:urine; D015813:Substance Abuse Detection / Q000379:methods
chemical_list: D020033:Protein Isoforms; D011994:Recombinant Proteins; D004921:Erythropoietin; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid
keywords: 2D electrophoresis; SDS-PAGE; desialylation; doping control; erythropoietin