Differentiation of endogenous erythropoietin and exogenous ESAs by Western blotting.
Differentiation of endogenous erythropoietin and exogenous ESAs by Western blotting.
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DOI：
10.1016/j.heliyon.2020.e05389
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发表时间：
2020-11
期刊：
影响因子：
4
通讯作者：
Nonoguchi H
中科院分区：
文献类型：
--
作者：
Yasuoka Y;Fukuyama T;Izumi Y;Yamashita T;Nakayama Y;Inoue H;Yanagita K;Oshima T;Yamazaki T;Uematsu T;Kobayashi N;Shimada Y;Nagaba Y;Mukoyama M;Sato Y;Sands JM;Kawahara K;Nonoguchi H
已经开发了非法使用红细胞生成刺激剂的兴奋剂检测方法。我们开发了一种新的蛋白质印迹法，用于检测和区分内源性促红细胞生成素（Epo，35-38 kDa）和外源性ESA（epoetin α和β，38-42 kDa;达依泊汀α，47-50 kDa; epoetin β pegol，93-110 kDa）。Epo和ESA是糖蛋白，使用肽-N-糖苷酶F的去糖基化将除epoetin β pegol外的所有Epo和ESA条带移动至22 kDa。我们从SDS-PAGE凝胶上切下Epo和ESA的条带，并通过液相色谱/质谱（LC/MS）对其进行分析。LC/MS检测到所有内源性Epo和外源性ESA均为去糖基化22 kDa Epo，表明LC/MS分析可确认Epo或ESA的存在，但无法区分内源性Epo和外源性ESA。我们提出了以下Epo兴奋剂测试：1）通过糖基化形式的蛋白质印迹法检测Epo或ESA; 2）通过去糖基化后的条带移动来增加可靠性;和3）通过使用切割凝胶的LC/MS分析来完全确认Epo或ESA。我们的方法的优点之一是在我们的Western印迹中不需要对Epo样品进行预纯化。生物技术;生物化学;分子生物学;公共卫生;血液系统;肾系统;红细胞生成素、红细胞生成刺激剂、蛋白质印迹法、液相色谱/质谱分析、掺杂、内源性、外源性。
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. Biotechnology; Biochemistry; Molecular Biology; Public Health; Hematological System; Renal System; erythropoietin, erythropoiesis-stimulating agents, Western blotting, Liquid Chromatography/Mass Spectrometry analysis, doping, endogenous, exogenous.
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影响因子：
2.9
作者：
Heuberger, Jules A. A. C.;van Eenoo, Peter;Cohen, Adam F.
通讯作者：
Cohen, Adam F.
影响因子：
64.8
作者：
Lasne, F;de Ceaurriz, J
通讯作者：
de Ceaurriz, J
DOI：
10.1007/978-1-61779-821-4_7
发表时间：
2012-01-01
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Reichel, Christian
通讯作者：
Reichel, Christian
影响因子：
4.4
作者：
Takahashi, Daisuke;Kawamura, Yukio;Uemura, Matsuo
通讯作者：
Uemura, Matsuo
影响因子：
4.3
作者：
Okano, Masato;Sato, Mitsuhiko;Kageyama, Shinji
通讯作者：
Kageyama, Shinji