Structural characterization of protein-protein interaction sites by FT-ICR MS

在六极杆离子导向器中，采用氢-氘（H/D）交换结合碰撞诱导解离（CID）技术，结合电喷雾离子化-傅立叶变换离子回旋共振质谱（ESI-FTICR MS）研究了半胱氨酸蛋白酶抑制剂与木瓜蛋白酶的相互作用。在木瓜蛋白酶存在或不存在的情况下，在不同的时间点分析氘掺入半胱氨酸蛋白酶抑制剂的骨架酰胺氢，检查由hexapole-CID产生的每个片段离子的质量。在半胱氨酸蛋白酶抑制剂-木瓜蛋白酶复合物的情况下，在整个半胱氨酸蛋白酶抑制剂序列中识别初始氘含量的显著下降。在数小时内识别出残基1 -10的区域中氘含量的显著降低，表明柔性N-末端区域应该已经用氢键紧密地固定在结合口袋中。这些结果 ...更多信息 这一结果与前人对半胱氨酸蛋白酶抑制剂的结构和抑制机制的研究结果一致。利用电喷雾质谱（ESI-FTICRMS）技术，在短时间内，以木瓜蛋白酶和半胱氨酸蛋白酶抑制剂N端部分滞后的复合物为研究对象，研究了N端部分滞后的复合物的电喷雾质谱。当半胱氨酸蛋白酶抑制剂与等摩尔量的木瓜蛋白酶混合时，游离全长半胱氨酸蛋白酶抑制剂的相对强度显著降低。这可能是由于完整的半胱氨酸蛋白酶抑制剂与木瓜蛋白酶的结合亲和力高于截短的半胱氨酸蛋白酶抑制剂。这些结果与半胱氨酸蛋白酶抑制剂-木瓜蛋白酶复合物H/D交换的结果一致。用同样的方法研究了肽蜂毒肽与脂质胶束十二烷基磷酸胆碱相互作用引起的结构变化。证实蜂毒素在脂质存在下处于稳定构象，而在脂质不存在下其结构非常灵活。少

The interaction between cystatin and papain was investigated by hydrogen-deuterium (H/D) exchange in conjunction with successive analysis by collision induced dissociation (CID) in a hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium incorporation into backbone amide hydrogens of cystatin was analyzed at different time points in the presence or absence of papain examining the mass of each fragment ion produced by hexapole-CID.In the absence of papain, amide hydrogens in short amino-terminal fragments were highly deuterated within 1min. In the case of cystatin-papain complex, significant drops in initial deuterium contents were recognized throughout the sequence of cystatin. Remarkable reduction in deuterium content in the region of residues l-10 was recognized for hours, suggesting that the flexible N-terminal region should have been tightly fixed in the binding pocket with hydrogen bonds. These resu … More lts were consistent with the previous studies on the structure and inhibition mechanism of cystatin. It was demonstrated that protein-protein interaction can be characterized by H/D exchange in combination with CID using ESI-FTICR MS within a short time using a small amount of sample.ESIMS was applied to the complex of papain and cystatin with partly lagged N-terminus. When cystatin was mixed with equimolar quantity of papain, the relative intensity of the free full-length cystatin dramatically decreased. It might be caused by the higher binding affinity of the intact cystatin for papain than those of the truncated forms. These results were consistent with those of the H/D exchange of cystatin-papain complex.The structural change of a peptide, melittin, caused by the interaction with lipid-micelles, dodecylphosphocholine, was investigated using the same method. It was confirmed that melittin is in a stable conformation in the presence of the lipid, while its structure is very flexible in the absence of the lipid. Less