Probing structures of large protein complexes using zero-length cross-linking.
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.
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.
Roland F
RF
Rivera-Santiago
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.
Sira
S
Sriswasdi
The Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA 19104, United States.
Sandra L
SL
Harper
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. Electronic address: speicher@wistar.org.
David W
DW
Speicher
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006801:Humans; D046911:Macromolecular Substances / Q000032:analysis* / Q000737:chemistry*; D011487:Protein Conformation; D053719:Tandem Mass Spectrometry / Q000379:methods*
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances
Chemical cross-linking; Cross-link identification; Mass spectrometry; Molecular modeling; Structure; Zero-length cross-linking