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首页> 外文期刊>European journal of mass spectrometry >Mapping protein interfaces by chemical cross-linking and Fourier transform ion cyclotron resonance mass spectrometry: application to a calmodulin/adenylyl cyclase 8 peptide complex
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Mapping protein interfaces by chemical cross-linking and Fourier transform ion cyclotron resonance mass spectrometry: application to a calmodulin/adenylyl cyclase 8 peptide complex

机译：通过化学交联和傅立叶变换离子回旋共振质谱分析蛋白质界面：在钙调蛋白/腺苷酸环化酶8肽复合物中的应用

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摘要

Chemical cross-linking-an established technique in protein chemistry-has re-emerged, in combination with mass spectrometric analysis of the reaction products, as a valuable tool to identify interacting amino acid sequences in protein complexes. In the present study, we are mapping the interface of the calcium-dependent complex between calmodulin (CaM) and a peptide derived from the C-terminal region of adenylyl cyclase 8 (AC 8). Cross-linking reactions are performed using the two amine-reactive, isotope-labeled (d(0) and d(4)) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS(2)G (bis[sulfosuccinimidyl]glutarate) as well as the "zero-length" cross-linker (EDC, ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride). After separation of the cross-linking reaction mixtures by one-dimensional gel electrophoresis (sodium dodecyl sulphate polyacrylamide gel) and in-gel digestion of the cross-linked complexes, the resulting peptide mixtures are analyzed by nano-high-performance liquid chromatographyano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. The identified intermolecular cross-linking products will give further insight into calmodulin/adenylyl cyclase 8 interaction.
机译：结合了反应产物的质谱分析，化学交联（一种蛋白质化学中的成熟技术）已经重新出现，成为鉴定蛋白质复合物中相互作用的氨基酸序列的有价值的工具。在本研究中，我们正在绘制钙调蛋白（CaM）与源自腺苷酸环化酶8（AC 8）C端区域的肽之间的钙依赖性复合物的界面图。交联反应使用两种胺反应性的，同位素标记的（d（0）和d（4））交联剂BS3（双[磺基琥珀酰亚胺基]硫酸根）和BS（2）G（双[磺基琥珀酰亚胺基]谷氨酸）和“零长度”交联剂（EDC，乙基-3- [3-二甲基氨基丙基]碳二亚胺盐酸盐）。通过一维凝胶电泳（十二烷基硫酸钠聚丙烯酰胺凝胶）分离交联反应混合物并在凝胶中消化交联的络合物后，通过纳米高效液相色谱/纳米分析所得的肽混合物-电喷雾电离傅里叶变换离子回旋共振质谱。鉴定出的分子间交联产物将进一步深入了解钙调蛋白/腺苷酸环化酶8的相互作用。

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