Quantifying Ligand Binding to Large Protein Complexes Using Electrospray Ionization Mass Spectrometry
Quantifying Ligand Binding to Large Protein Complexes Using Electrospray Ionization Mass Spectrometry
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DOI：
10.1021/ac3005082
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发表时间：
2012-05-01
影响因子：
7.4
通讯作者：
Klassen, John S.
中科院分区：
文献类型：
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作者：
El-Hawiet, Amr;Kitova, Elena N.;Klassen, John S.
描述了一种用于定量蛋白质-配体复合物的电喷雾电离质谱（ESI-MS）方法，该蛋白质-配体复合物不能通过ESI-MS直接检测。代理蛋白质ESI-MS方法将直接ESI-MS结合测量与竞争性蛋白质-配体结合结合相结合。为了实施该方法，使用代理蛋白（P-代理）来定量监测配体与目标蛋白结合的程度，所述代理蛋白以已知的亲和力与目标配体特异性相互作用并且可以通过ESI-MS直接检测。建立了一个数学框架的关联常数（K-a）的蛋白质-配体结合的代理蛋白质ESI-MS方法，实现了一个P-代理含有一个单一的配体结合位点，给出。还描述了代理蛋白质ESI-MS方法的修改形式，该方法考虑配体浓度的实时变化。对于噬菌体P22的180 kDa野生型同源三聚体尾刺蛋白及其内鼠李糖苷酶点突变体（D392 N）与其配体之间的相互作用，证明了这些方法的可靠性，所述配体包含来自鼠伤寒沙门氏菌血清型的两个和三个O-抗原重复：八糖（[α-Gal-（1 -> 2）-[α-Abe-（1 -> 3）]-α-Man-（1 -> 4）-α-Rha]（2））和十二糖（[α-Gal-（1 -> 2）-[α-Abe-（1 -> 3）]-α-Man-（1 -> 4）-α-Rha]（3））。与两种配体结合的27 kDa单链抗体用作P-代理。在10 ℃和25 ℃下进行的结合测量的结果与先前使用荧光猝灭测定法测量的K-a值非常一致。
An electrospray ionization mass spectrometry (ESI-MS) method for quantifying protein-ligand complexes that cannot be directly detected by ESI-MS is described. The proxy protein ESI-MS method combines direct ESI-MS binding measurements with competitive protein-ligand binding. To implement the method, a proxy protein (P-proxy), which interacts specifically with the ligand of interest with known affinity and can be detected directly by ESI-MS, is used to quantitatively monitor the extent of ligand binding to the protein of interest. A mathematical framework for establishing the association constant (K-a) for protein-ligand binding by the proxy protein ESI-MS method, implemented with a P-proxy containing a single ligand binding site, is given. A modified form of the proxy protein ESI-MS method, which accounts for real-time changes in ligand concentration, is also described. The reliability of these methods is demonstrated for the interactions between the 180 kDa wildtype homotrimeric tailspike protein of the bacteriophage P22 and its endorhamnosidase point mutant (D392N) with its ligands comprising two and three O-antigen repeats from Salmonella enterica serovar Typhimurium: octasaccharide ([alpha-Gal-(1 -> 2)-[alpha-Abe-(1 -> 3)]-alpha-Man-(1 -> 4)-alpha-Rha](2)) and dodecasaccharide ([alpha-Gal-(1 -> 2)-[alpha-Abe-(1 -> 3)]-alpha-Man-(1 -> 4)-alpha-Rha](3)). A 27 kDa single chain antibody, which binds to both ligands, served as P-proxy. The results of binding measurements performed at 10 and 25 degrees C are in excellent agreement with K-a values measured previously using a fluorescence quenching assay.