Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle (Saale), Germany.
Anal Chem. 2010 Aug 15;82(16):6958-68. doi: 10.1021/ac101241t.
Chemical cross-linking combined with a subsequent enzymatic cleavage of the created cross-linked complex and a mass spectrometric analysis of the resulting cross-linked 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 alpha, 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.
化学交联结合随后的酶切交联复合物，并对产生的交联肽混合物进行质谱分析，这为获得低分辨率蛋白质结构并深入了解蛋白质界面提供了一种替代高分辨率分析（如 NMR 光谱学或 X 射线晶体学）的方法。在这里，我们描述了一种新的基于尿素的交联剂，它允许通过基于特征产物离子和恒定中性损失的碰撞诱导解离 (CID) 串联 MS 实验来区分不同的交联产物。该新型交联剂是我们正在努力开发的用于分析交联蛋白质和蛋白质复合物的碰撞诱导解离试剂的一部分。我们的创新分析概念以 Munc13-1 肽和重组表达的过氧化物酶体增殖物激活受体 α 的配体结合结构域为例，通过离线纳升 HPLC/MALDI-TOF/TOF 质谱和在线纳升 HPLC/纳升电喷雾-LTQ-轨道阱质谱分析了交联反应混合物。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鉴定不同交联物种（即修饰肽以及肽内和肽间交联）的过程，并大大降低了鉴定假阳性交联的可能性。我们的新型基于尿素的 CID 可裂解交联剂有望在自动化分析蛋白质 3D 结构和蛋白质-蛋白质复合物方面具有很高的优势。
