Mapping Protein Interfaces by Chemical Cross-Linking and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Application to a Calmodulin/Adenylyl Cyclase 8 Peptide Complex
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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DOI：
10.1255/ejms.748
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发表时间：
2005-10
影响因子：
1.3
通讯作者：
A. Schmidt;S. Kalkhof;C. Ihling;D. Cooper;Andrea Sinza
中科院分区：
文献类型：
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作者：
A. Schmidt;S. Kalkhof;C. Ihling;D. Cooper;Andrea Sinza
化学交联-蛋白质化学中的一项成熟技术-重新出现，与反应产物的质谱分析相结合，作为鉴定蛋白质复合物中相互作用氨基酸序列的有价值的工具。在目前的研究中，我们绘制了钙调蛋白（CaM）和腺苷酸环化酶8 (ac8) c端区衍生的肽之间钙依赖性复合物的界面。交联反应使用两种胺反应，同位素标记（d0和d4）交联剂BS3（双[亚砜丁二酰]亚硫酸盐）和BS2G（双[亚砜丁二酰]戊二酸酯）以及“零长度”交联剂（EDC，乙基-3-[3-二甲氨基丙基]盐酸碳二酰亚胺）进行。通过一维凝胶电泳（十二烷基硫酸钠聚丙烯酰胺凝胶）对交联反应混合物进行分离，并对交联配合物进行凝胶内消化后，用纳米高效液相色谱/纳米电喷雾电离傅里叶变换离子回旋共振质谱分析得到的肽混合物。鉴定的分子间交联产物将进一步深入了解钙调素/腺苷酸环化酶8的相互作用。
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 (d0 and d4) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS2G (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 chromatography/nano-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.