Reichel Christian
Doping Control Laboratory, Austrian Research Centers GmbH-ARC, A-2444 Seibersdorf, Austria.
J Mass Spectrom. 2008 Jul;43(7):916-23. doi: 10.1002/jms.1444.
The detection of doping with recombinant erythropoietins (Epo) by isoelectric focusing (IEF) and Western double blotting strongly relies on the specificity of the detection antibody used. Currently a monoclonal mouse antibody (clone AE7A5) is used for that purpose. Despite its excellent sensitivity (amol range) the antibody shows some nonspecific binding behavior. However, the binding occurs outside the currently used pH range for evaluating erythropoietin IEF profiles. A shotgun proteomics approach is described consisting of preparative IEF on large-sized carrier ampholyte gels (pH 3-5), SDS-PAGE, Western single and double blotting, on-membrane elution of intact proteins, on-membrane and in-solution tryptic digestions, as well as nano-HPLC peptide separation and high-resolution high-mass accuracy ESI-MS/MS peptide sequencing. The nonspecifically interacting protein could be identified as zinc-alpha-2-glycoprotein (ZAG). Confirmation analyses were performed using recombinant ZAG (rhZAG) and a monoclonal anti-ZAG antibody. It could be demonstrated that the binding of the monoclonal antihuman EPO antibody (clone AE7A5) to ZAG occurs in a highly concentration-dependant manner and that only samples containing increased amounts of urinary ZAG lead to a detectable interaction of the AE7A5 antibody on Epo-IEF gels.
通过等电聚焦(IEF)和蛋白质免疫印迹法检测重组促红细胞生成素(Epo)兴奋剂依赖于所用检测抗体的特异性。目前使用一种单克隆小鼠抗体(克隆AE7A5)进行此项检测。尽管该抗体具有出色的灵敏度(飞摩尔范围),但仍表现出一些非特异性结合行为。然而,这种结合发生在当前用于评估促红细胞生成素IEF图谱的pH范围之外。本文描述了一种鸟枪法蛋白质组学方法,包括在大型载体两性电解质凝胶(pH 3 - 5)上进行制备性IEF、SDS - PAGE、蛋白质免疫单印迹和双印迹、完整蛋白质的膜上洗脱、膜上和溶液中的胰蛋白酶消化,以及纳米HPLC肽段分离和高分辨率高精度ESI - MS/MS肽段测序。非特异性相互作用的蛋白质被鉴定为锌α2糖蛋白(ZAG)。使用重组ZAG(rhZAG)和单克隆抗ZAG抗体进行了确认分析。结果表明,单克隆抗人EPO抗体(克隆AE7A5)与ZAG的结合以高度浓度依赖性方式发生,并且只有尿液中ZAG含量增加的样本才会导致AE7A5抗体在Epo - IEF凝胶上产生可检测到的相互作用。