Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations
Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations
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DOI：
10.1073/pnas.1318405111
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发表时间：
2014-02-11
影响因子：
11.1
通讯作者：
Landes, Christy F.
中科院分区：
文献类型：
--
作者：
Kisley, Lydia;Chen, Jixin;Landes, Christy F.
色谱蛋白质分离、免疫测定和生物传感通常都涉及蛋白质吸附到用带电、疏水或亲和配体修饰的表面。尽管在整个制药行业中使用越来越广泛，但关于蛋白质与单个色谱吸附剂位点相互作用的机理细节仅可通过从系综测量（例如结合等温线、量热法和色谱法）进行推断来获得。在这项工作中，我们提出了直接的超分辨率映射和动力学特性的功能位点的离子交换配体的基础上琼脂糖，蛋白质色谱中常用的支持基质。通过量化单个蛋白质与单个带电配体的相互作用，我们证明了电荷簇对于创建可检测的吸附位点是必要的，并且即使化学性质相同的配体也会创建依赖于界面处的空间可用性的不同动力学性质的吸附位点。此外，我们与实验结果的随机理论色谱。从分子尺度数据计算的模拟洗脱曲线表明，如果有可能将均匀的最佳相互作用设计成离子交换系统，则可以通过故意利用目前仅偶然主导离子交换过程的群集相互作用来将分离效率提高多达五倍。
Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.