2020年11月
Differentiation of endogenous erythropoietin and exogenous ESAs by Western blotting.
Heliyon
ダウンロード
回数 : 66
- 巻
- 6
- 号
- 11
- 開始ページ
- e05389
- 終了ページ
- e05389
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/j.heliyon.2020.e05389
- 出版者・発行元
- Elsevier BV
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.
- リンク情報
- ID情報
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- DOI : 10.1016/j.heliyon.2020.e05389
- ISSN : 2405-8440
- PubMed ID : 33195841
- PubMed Central 記事ID : PMC7644904