Hydrogen/deuterium exchange- and protease digestion-based screening assay for protein-ligand binding detection.
Hydrogen/deuterium exchange- and protease digestion-based screening assay for protein-ligand binding detection.
复制标题
基于氢/氘交换和蛋白酶消化的筛选测定，用于蛋白质-配体结合检测。
DOI：
10.1021/ac900854t
复制
发表时间：
2009
影响因子：
7.4
通讯作者：
Fitzgerald,MichaelC
中科院分区：
文献类型：
--
作者：
Hopper,ErinD;Pittman,AdrianneMC;Tucker,ChandraL;Campa,MichaelJ;PatzJr,EdwardF;Fitzgerald,MichaelC
根据 H/D 交换率 (SUPREX)，将蛋白酶消化策略纳入未纯化蛋白质的单点稳定性中，这是一种基于氢/氘 (H/D) 交换和质谱分析的检测方法，用于检测蛋白质-配体结合。单点 SUPREX 是 SUPREX 的缩写形式，其中通过测量配体结合后蛋白质热力学稳定性的增加来检测蛋白质-配体结合相互作用。新的蛋白酶消化方案显着提高了单点 SUPREX 的效率，因为在单点 SUPREX 的基质辅助激光解吸电离 (MALDI) 读数中，可以比完整蛋白质质量更精确地确定肽质量。该方案在两个模型蛋白质系统的测试筛选中进行了评估，包括亲环蛋白 A (CypA) 和人丙氨酸：乙醛酸转氨酶 (AGTmi) 的次要等位基因变体。对两种蛋白质获得的测试筛选结果表明，在区分命中和非命中时，单点 SUPREX 蛋白酶消化方案的肽读数比原始单点 SUPREX 方案的完整蛋白质读数更有效。除了筛选效率的提高之外，本文描述的蛋白酶消化策略预计将显着提高单点 SUPREX 测定的通用性。
A protease digestion strategy was incorporated into single-point stability of unpurified proteins from rates of H/D exchange (SUPREX), which is a hydrogen/deuterium (H/D) exchange- and mass spectrometry-based assay for the detection of protein−ligand binding. Single-point SUPREX is an abbreviated form of SUPREX in which protein−ligand binding interactions are detected by measuring the increase in a protein’s thermodynamic stability upon ligand binding. The new protease digestion protocol provides a noteworthy increase in the efficiency of single-point SUPREX because peptide masses can be determined with greater precision than intact protein masses in the matrix-assisted laser desorption ionization (MALDI) readout of single-point SUPREX. The protocol was evaluated in test screens on two model protein systems, including cyclophilin A (CypA) and the minor allele variant of human alanine:glyoxylate aminotransferase (AGTmi). The test screening results obtained on both proteins revealed that the peptide readout of the single-point SUPREX-protease digestion protocol was more efficient than the intact protein readout of the original single-point SUPREX protocol at discriminating hits and nonhits. In addition to this improvement in screening efficiency, the protease digestion strategy described here is expected to significantly increase the generality of the single-point SUPREX assay.