Cleavable Cross-Linker for Protein Structure Analysis: Reliable Identification of Cross-Linking Products by Tandem MS
Cleavable Cross-Linker for Protein Structure Analysis: Reliable Identification of Cross-Linking Products by Tandem MS
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DOI：
10.1021/ac101241t
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发表时间：
2010-08-15
影响因子：
7.4
通讯作者：
Sinz, Andrea
中科院分区：
文献类型：
--
作者：
Mueller, Mathias Q.;Dreiocker, Frank;Sinz, Andrea
化学交联与随后产生的交联复合体的酶裂解相结合，并对所产生的交联肽混合物进行质谱分析，为高分辨率分析提供了另一种方法，例如核磁共振光谱或X射线结晶学，以获得低分辨率的蛋白质结构和洞察蛋白质界面。在这里，我们描述了一种新型的尿基交联剂，它可以根据特征产物离子和恒定的中性损失，通过碰撞诱导解离(CID)串联MS实验来区分不同的交联物。这种新型交联剂是我们正在进行的开发碰撞诱导解离试剂的努力的一部分，这种试剂可以有效地分析交联的蛋白质和蛋白质复合体。我们的创新分析概念以Munc13-1多肽和重组表达的过氧化物酶体增殖物激活受体a的配体结合域为例，对其交联反应混合物进行了离线纳米高效液相色谱/MALDI-TOF/TOF质谱分析和在线纳米高效液相色谱/纳米ESI-LTQ-Orbitrap质谱仪分析。这种新型交联剂的特征片段离子模式极大地简化了从复杂混合物中识别不同的交联物，即修饰肽以及肽内和肽间交联物，并极大地降低了识别假阳性交联物的可能性。我们的新型基于尿素的CID可裂解交联剂有望在以自动化方式分析蛋白质3D结构和蛋白质-蛋白质复合体方面具有极大的优势。
Chemical cross-linking combined with a subsequent enzymatic cleavage of the created cross-linked complex and a mass spectrometric analysis of the resulting crosslinked peptide mixture presents an alternative approach to high-resolution analysis, such as NMR spectroscopy or X-ray crystallography, to obtain low-resolution protein structures and to gain insight into protein interfaces. Here, we describe a novel urea-based cross-linker, which allows distinguishing different cross-linking products by collision-induced dissociation (CID) tandem MS experiments based on characteristic product ions and constant neutral losses. The novel cross-linker is part of our ongoing efforts in developing collision-induced dissociative reagents that allow an efficient analysis of cross-linked proteins and protein complexes. Our innovative analytical concept is exemplified for the Munc13-1 peptide and the recombinantly expressed ligand binding domain of the peroxisome proliferator-activated receptor a, for which cross-linking reaction mixtures were analyzed both by offline nano-HPLC/MALDI-TOF/TOF mass spectrometry and by online nano-HPLC/nano-ESI-LTQ-orbitrap mass spectrometry. The characteristic fragment ion patterns of the novel cross-linker greatly simplify the identification of different cross-linked species, namely, modified peptides as well as intrapeptide and interpeptide cross-links, from complex mixtures and drastically reduce the potential of identifying false-positive cross-links. Our novel urea-based CID cleavable cross-linker is expected to be highly advantageous for analyzing protein 3D structures and protein-protein complexes in an automated manner.