Rivera-Santiago Roland F, Sriswasdi Sira, Harper Sandra L, Speicher David W
The Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA 19104, United States; Biochemistry and Molecular Biophysics Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, United States.
The Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA 19104, United States; Department of Biological Sciences, Graduate School of Sciences, The University of Tokyo, Tokyo 113-0032, Japan.
Methods. 2015 Nov 1;89:99-111. doi: 10.1016/j.ymeth.2015.04.031. Epub 2015 May 1.
Structural mass spectrometry (MS) is a field with growing applicability for addressing complex biophysical questions regarding proteins and protein complexes. One of the major structural MS approaches involves the use of chemical cross-linking coupled with MS analysis (CX-MS) to identify proximal sites within macromolecules. Identified cross-linked sites can be used to probe novel protein-protein interactions or the derived distance constraints can be used to verify and refine molecular models. This review focuses on recent advances of "zero-length" cross-linking. Zero-length cross-linking reagents do not add any atoms to the cross-linked species due to the lack of a spacer arm. This provides a major advantage in the form of providing more precise distance constraints as the cross-linkable groups must be within salt bridge distances in order to react. However, identification of cross-linked peptides using these reagents presents unique challenges. We discuss recent efforts by our group to minimize these challenges by using multiple cycles of LC-MS/MS analysis and software specifically developed and optimized for identification of zero-length cross-linked peptides. Representative data utilizing our current protocol are presented and discussed.
结构质谱（MS）是一个在解决有关蛋白质和蛋白质复合物的复杂生物物理问题方面适用性不断增长的领域。主要的结构质谱方法之一涉及使用化学交联结合质谱分析（CX-MS）来识别大分子内的近端位点。识别出的交联位点可用于探测新的蛋白质-蛋白质相互作用，或者所得到的距离限制可用于验证和完善分子模型。本综述聚焦于“零长度”交联的最新进展。零长度交联试剂由于缺乏间隔臂，不会向交联产物添加任何原子。这在提供更精确的距离限制方面具有主要优势，因为可交联基团必须处于盐桥距离内才能发生反应。然而，使用这些试剂鉴定交联肽存在独特的挑战。我们讨论了我们团队最近通过使用多个液相色谱-串联质谱（LC-MS/MS）分析循环以及专门为鉴定零长度交联肽而开发和优化的软件来尽量减少这些挑战的努力。展示并讨论了利用我们当前方案的代表性数据。
