Heterobifunctional isotope-labeled amine-reactive photo-cross-linker for structural investigation of proteins by matrix-assisted laser desorption/ionization tandem time-of-flight and electrospray ionization LTQ-Orbitrap mass spectrometry
Heterobifunctional isotope-labeled amine-reactive photo-cross-linker for structural investigation of proteins by matrix-assisted laser desorption/ionization tandem time-of-flight and electrospray ionization LTQ-Orbitrap mass spectrometry
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DOI：
10.1002/rcm.4188
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发表时间：
2009-09-01
影响因子：
2
通讯作者：
Sinz, Andrea
中科院分区：
文献类型：
--
作者：
Krauth, Fabian;Ihling, Christian H.;Sinz, Andrea
化学交联结合随后的酶消化和质谱分析所创建的交联产物提供了一种评估低分辨率蛋白质结构的替代方法。通过共价连接蛋白质或蛋白质复合物内的功能基团对，建立了一套结构上确定的相互作用。合成了异双官能团胺反应性光交联剂n-琥珀酰对苯甲酰二氢肉桂酸酯，作为非氘化衍生物（SBC）和双氘化衍生物（SBDC）。采用1:1的SBC和SBDC混合物进行交联实验，有助于根据片段离子的特征同位素模式在质谱中识别交联氨基酸。将交联剂应用于钙结合蛋白钙调素，随后采用纳米高效液相色谱-基质辅助激光解吸/电离串联飞行时间质谱（nano-HPLC/MALDI-TOF/TOF-MS）和纳米高效液相色谱/纳米电喷雾电离(ESI)-LTQ-Orbitrap-NlS对交联产物进行分析。版权所有John Wiley & Sons， Ltd. 2009
Chemical cross-linking combined with a subsequent enzymatic digestion and mass spectrometric analysis of the created cross-linked products presents an alternative approach to assess low-resolution protein structures. By covalently connecting pairs of functional groups within a protein or a protein complex a set of structurally defined interactions is built up. We synthesized the heterobifunctional amine-reactive photo-cross-linker N-succinimidyl p-benzoyldihydrocinnamate as a non-deuterated (SBC) and doubly deuterated derivative (SBDC). Applying a 1:1 mixture of SBC and SBDC for cross-linking experiments aided the identification of cross-linked amino acids in the mass spectra based on the characteristic isotope patterns of fragment ions. The cross-linker was applied to the calcium-binding protein calmodulin with a subsequent analysis of cross-linked products by nano-high-performance liquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (nano-HPLC/MALDI-TOF/TOF-MS) and nano-HPLC/nano-electrospray ionization (ESI)-LTQ-Orbitrap-NlS. Copyright (C) 2009 John Wiley & Sons, Ltd.