Top-down ESI-ECD-FT-ICR mass spectrometry localizes noncovalent protein-ligand binding sites
Top-down ESI-ECD-FT-ICR mass spectrometry localizes noncovalent protein-ligand binding sites
复制标题
DOI：
10.1021/ja063197p
复制
发表时间：
2006-11-15
影响因子：
15
通讯作者：
Loo, Joseph A.
中科院分区：
文献类型：
--
作者：
Xie, Yongming;Zhang, Jennifer;Loo, Joseph A.
电喷雾离子化（ESI）质谱（MS）能够测量和检测非共价蛋白质-配体和蛋白质-蛋白质复合物。然而，配体结合位点的信息不容易通过ESI-MS方法获得。电子捕获解离（ECD）有利于蛋白质分子的共价骨架键的切割。我们发现，ECD的这种特性转化为非共价蛋白质-配体复合物，因为蛋白质复合物的共价骨架键被解离，但非共价配体相互作用被保留。对于由140个残基、14.5 kDa α-突触核蛋白和一分子聚阳离子精胺（202 Da）形成的复合物，ECD产生保留蛋白-精胺非共价相互作用的产物离子。精胺结合定位于残基106−138; ECD数据与之前的溶液NMR研究一致。我们的研究表明，ECD质谱可以用来直接确定配体结合蛋白质靶点的网站。
Mass spectrometry (MS) with electrospray ionization (ESI) has the capability to measure and detect noncovalent protein−ligand and protein−protein complexes. However, information on the sites of ligand binding is not easily obtained by the ESI-MS methodology. Electron capture dissociation (ECD) favors cleavage of covalent backbone bonds of protein molecules. We show that this characteristic of ECD translates to noncovalent protein−ligand complexes, as covalent backbone bonds of protein complexes are dissociated, but the noncovalent ligand interaction is retained. For the complex formed from 140-residue, 14.5 kDa α-synuclein protein, and one molecule of polycationic spermine (202 Da), ECD generates product ions that retain the protein−spermine noncovalent interaction. Spermine binding is localized to residues 106−138; the ECD data are consistent with previous solution NMR studies. Our studies suggest that ECD mass spectrometry can be used to determine directly the sites of ligand binding to protein targets.