Affinity analysis of a protein-aptamer complex using nonequilibrium capillary electrophoresis of equilibrium mixtures
Affinity analysis of a protein-aptamer complex using nonequilibrium capillary electrophoresis of equilibrium mixtures
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DOI：
10.1021/ac026214b
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发表时间：
2003-03-15
影响因子：
7.4
通讯作者：
Krylov, SN
中科院分区：
文献类型：
--
作者：
Berezovski, M;Nutiu, R;Krylov, SN
我们提出了一种新的方法，允许使用低亲和力适配子作为亲和力探针用于蛋白质的定量分析。该方法基于蛋白质及其荧光标记适体的平衡混合物(NECEEM)的非平衡毛细管电泳法。一般来说，带有荧光标记适体的蛋白质的NECEEM产生具有三个特征的电泳图：两个峰和一条指数曲线。两个峰对应于(I)平衡混合物中游离适配子的平衡量和(Ii)在检测时保持不变的蛋白质-适配子复合体的量。指数部分归因于非平衡条件下分离过程中的络合物衰变。在已知蛋白质浓度的实验中，对这三个特征的简单分析可用于测定适体-蛋白质复合体的平衡解离常数K-d。在未知蛋白质浓度和已知Kd值的实验中，对这三个特征的类似分析允许确定蛋白质浓度。在这项原理证明工作中，NECEEM方法被应用于在蛋白质-适配子复合体在分离过程中完全腐烂的条件下，使用荧光素标记的适配子来分析凝血酶。我们证明，尽管有衰变，NECEEM可以在不牺牲准确性的情况下检测到低至4x10(6)个蛋白质分子。这种灵敏度与其他人报道的基于适配子的平衡法相当。因此，所提出的基于NECEEM的方法允许使用适配子进行高度敏感的蛋白质亲和力分析，即使在蛋白质-适配子复合体不稳定的情况下也是如此。
We propose a new method that allows the use of low-affinity aptamers as affinity probes in quantitative analyses of proteins. The method is based on nonequilibrium capillary electrophoresis of the equilibrium mixture (NECEEM) of a protein with its fluorescently labeled aptamer. In general, NECEEM of a protein with a fluorescently labeled aptamer generates an electropherogram with three characteristic features: two peaks and an exponential curve. Two peaks correspond to (i) the equilibrium amount of free aptamer in the equilibrium mixture and (ii) the amount of the protein-aptamer complex that remains intact at the time of detection. The exponential part is ascribed to the complex decaying during separation under nonequilibrium conditions. Simple analysis of the three features in experiments with known concentrations of the protein can be used for the determination of the equilibrium dissociation constant, K-d, of the aptamer-protein complex. Similar analysis of the three features in the experiment with unknown concentration of the protein and known Kd value allows the determination of the protein concentration. In this proof-of-principle work, the NECEEM method was applied to the analysis of thrombin using a fluorescein-labeled aptamer under the conditions at which the protein-aptamer complex completely decayed during the separation. We demonstrated that, despite the decay, as few as 4 x 10(6) molecules of the protein could be detected with NECEEM without sacrificing the accuracy. This sensitivity is comparable with that reported by others for the aptamer-based equilibrium method. Thus, the proposed NECEEM-based method allows the use of aptamers for highly sensitive affinity analysis of proteins even when protein-aptamer complexes are unstable.