Jonker Niels, Lingeman Henk, Irth Hubertus
BioMolecular Analysis Group, Department of Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Chromatographia. 2010 Jul;72(1-2):7-13. doi: 10.1365/s10337-010-1586-x. Epub 2010 May 23.
A new methodology is described enabling the affinity screening of potential ligands towards the human estrogen receptor alpha ligand binding domain (ERalpha-LBD). In-solution incubation is performed of the analyte and the His-tagged ERalpha-LBD. The bound complex is immobilized on a nickel-loaded protein-affinity selection column, where after the unbound fraction is removed. The immobilized protein-ligand complex is exposed to a decreased pH value and an increased organic modifier concentration releasing the ligand for MS detection, and precipitating the proteins on a filter positioned between the affinity column and the mass spectrometer. The trapping column can be regenerated for reuse at least 70 times. The advantages of the methodology over existing methodologies are the absence of a pre-concentration as well as a chromatographic separation step, resulting in a significantly shorter analysis time compared to previously described procedures, and in addition, allowing the determination of solutes with unfavorable chromatographic properties. The overall analysis time now can be reduced about 250% to approximately 6 min. Replacing the filters after every measurement results in an intra-day standard deviation of 14.8% and an inter-day standard deviation of 21.3%.
本文描述了一种新方法，可用于对潜在配体与人雌激素受体α配体结合域（ERα-LBD）进行亲和筛选。将分析物与His标签的ERα-LBD进行溶液孵育。结合的复合物固定在负载镍的蛋白质亲和选择柱上，然后去除未结合部分。将固定化的蛋白质-配体复合物置于降低的pH值和增加的有机改性剂浓度条件下，释放配体用于质谱检测，并使蛋白质沉淀在位于亲和柱和质谱仪之间的滤膜上。捕集柱可至少再生70次以供重复使用。该方法相对于现有方法的优点在于无需预浓缩以及色谱分离步骤，与先前描述的方法相比，分析时间显著缩短，此外，还能测定具有不良色谱性质的溶质。现在总的分析时间可减少约至250%，约为6分钟。每次测量后更换滤膜，日内标准偏差为14.8%，日间标准偏差为21.3%。
