PEPTIDES 2019 CXMS Analysis for Complex Protein Structures and Protein-Protein Interactions (#58)
Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) has emerged as a powerful tool for studying protein complex structures and protein-protein interactions. However, CXMS is limited by the low abundance of cross-linked peptides and high sample complexity. Here, we designed and synthesized a series of novel Lysine-targeted enrichable cross-linker (Leiker) molecules containing a biotin tag for affinity purification, a chemical cleavage site that releases cross-links without carrying the biotin or other unnecessary structures for LC-MS/MS, and an isotopic tag for quantitative CXMS analysis. We found the optimal Leiker design and demonstrate that it effectively facilitates CXMS analysis of a few samples of high complexity, including purified macromolecular machinery, crude immunoprecipitates, and whole-cell lysates. Leiker thus enables comprehensive analysis of large protein complexes and facilitates the construction of direct protein-protein interaction networks. In particular, Leiker enabled identification of more than 3000 non-redundant inter-linked sites in E. coli whole-cell lysates, significantly improving the breadth of proteome-wide CXMS analysis. Finally, by revealing the RNA binding sites of a protein, we show that Leiker-assisted quantitative CXMS can be used to probe conformational changes of a protein or a protein complex.
- Tan, D.; Li, Q.; Zhang, M.; Liu, C.; Ma, C.; Zhang, P.; Ding, Y.; Fan, S.; Tao, L.; Yang, B.; Li, X.; Ma, S.; Liu, J.; Feng, B.; Liu, X.; Wang, H.; He, S.; Gao, N.; Ye, K.; Dong, M.*; Lei, X.* “Trifunctional Cross-Linker for Mapping Protein-Protein Interaction Networks and Comparing Protein Conformational States” eLife 2016; 5:e12509.
