Differentiation of endogenous erythropoietin and exogenous ESAs by Western blotting

• Authors: Yukiko Yasuoka, Kitasato University; Takashi Fukuyama, Kitasato University; Yuichiro Izumi, Kumamoto University; Tetsuro Yamashita, Iwate University; Yushi Nakayama, Kumamoto University; Hideki Inoue, Kumamoto University; Kengo Yanagita, Kitasato University; Tomomi Oshima, Kitasato University; Taiga Yamazaki, Kitasato University; Takayuki Uematsu, Kitasato University; Noritada Kobayashi, Kitasato University; Yoshitaka Shimada, Kitasato University; Yasushi Nagaba, Kitasato University; Masashi Mukoyama, Kumamoto University; Yuichi Sato, Kitasato University; Jeff Sands, Emory University; Katsumasa Kawahara, Kitasato University; Hiroshi Nonoguchi, Kitasato University
• Journal Title: HELIYON
• Volume: Volume 6, Number 11
• Publisher: ELSEVIER SCI LTD | 2020-11-01, Pages e05389-e05389
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1016/j.heliyon.2020.e05389
• Subjects: Biology, Molecular; Chemistry, Biochemistry; Health Sciences, Medicine and Surgery
• Keywords: Science & Technology; Multidisciplinary Sciences; Science & Technology - Other Topics; Biotechnology; Biochemistry; Molecular biology; Public health; Hematological system; Renal system; Erythropoietin; Erythropoiesis-stimulating agents; Western blotting; Liquid chromatography/mass spectrometry analysis; Doping; Endogenous; Exogenous; URINE

Abstract

Doping tests for the illegal use of erythropoiesis-stimulating agents (ESAs) have been developed. We developed a new Western blotting method to detect and distinguish endogenous erythropoietin (Epo, 35-38 kDa) and exogenous ESAs (epoetin α and β, 38-42 kDa; darbepoetin α, 47-50 kDa; epoetin β pegol, 93-110 kDa). Epo and ESAs are glycoproteins and deglycosylation using peptide-N-glycosidase F shifted all Epo and ESA bands except epoetin β pegol to 22 kDa. We cut the bands of Epo and ESAs from SDS-PAGE gels and analyzed them by Liquid Chromatography/Mass Spectrometry (LC/MS). LC/MS detected all endogenous Epo and exogenous ESAs as deglycosylated 22 kDa Epo, indicating that LC/MS analysis could confirm the presence of Epo or ESA, but could not distinguish between endogenous Epo and exogenous ESAs. We propose the following Epo doping tests: 1) detect Epo or ESAs by Western blotting of the glycosylated form; 2) increase the reliability by the band shift following deglycosylation; and 3) complete confirmation of Epo or ESA by LC/MS analysis using cut gels. One of the advantages of our method is that pre-purification of samples for Epo is not required in our Western blotting.