Summary
Most proteins function through protein complex assemblies. Defining and mapping protein complex networks are crucial elements in the fundamental understanding of biological processes. The ability to measure protein–protein interactions in biological systems has undergone significant advances in the past decade due to emergence and growth of numerous new molecular biology and mass spectrometry technologies. Chemical cross-linking, along with yeast two-hybrid, fluorescence resonant energy transfer (FRET), and co-immunoprecipitation have become important tools for detection and characterization of protein–protein interactions. Individual protein members in a noncovalent complex assembly remain in close proximity which is within the reach of the two reactive groups of a cross-linker. Thus cross-linking reactions have potential for linking two interacting proteins which exist in close proximity. In general, chemical cross-linking experiments are carried out by first linking the interacting proteins through covalent bonds followed by a series of well-established protocols–SDS-PAGE, in-gel digestion, and shotgun LC/ MS/MS for identification of the cross-linked proteins. These approaches have been employed for both mapping topology of protein complex in vitro and determining the protein interaction partners in vivo.
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Back, J. W., de Jong, L., Muijsers, A. O., and de Koster, C. G. (2003) Chemical cross-linking and mass spectrometry for protein structural modeling. J Mol Biol 331, 303–13.
Trakselis, M. A., Alley, S. C., and Ishmael, F. T. (2005) Identification and mapping of protein–protein interactions by a combination of cross-linking, cleavage, and proteomics. Bioconjugate Chem 16, 741–50.
Sinz, A. (2006) Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein–protein interactions. Mass Spectrom Rev 25, 663–82.
Vasilescu, J., and Figeys, D. (2006) Mapping protein–protein interactions by mass spectrometry. Curr Opin Biotechnol 17, 394–9.
Trester-Zedlitz, M., Kamada, K., Burley, S. K., Fenyo, D., Chait, B. T., and Muir, T. W. (2003) A modular cross-linking approach for exploring protein interactions. J Am Chem Soc 125, 2416–25.
Chang, Z., Kuchar, J., and Hausinger, R. P. (2004) Chemical cross-linking and mass spectrometric identification of sites of interaction for UreD, UreF, and urease. J Biol Chem 279, 15305–13.
Petrotchenko, E. V., Olkhovik, V. K., and Borchers, C. H. (2005) Isotopically coded cleavable cross-linker for studying protein–protein interaction and protein complexes. Mol Cell Proteomics 4, 1167–79.
Kalkhof, S., Ihling, C., Mechtler, K., and Sinz, A. (2005) Chemical cross-linking and high-performance fourier transform ion cyclotron resonance mass spectrometry for protein interaction analysis: Application to a calmodulin/target peptide complex. Anal Chem 77, 495–503.
Tang, X., Munske, G. R., Siems, W. F., and Bruce, J. E. (2005) Mass spectrometry identifiable cross-linking strategy for studying protein–protein interactions. Anal Chem 77, 311–8.
Chen, Y., Ebright, Y. W., and Ebright, R. H. (1994) Identification of the target of a tran- scription activator protein by protein–protein photocrosslinking. Science 265, 90–2.
Wells, J., and Farnham, P. J. (2002) Characterizing transcription factor binding sites using formaldehyde crosslinking and immu-noprecipitation. Methods26, 48–56.
Schmitt-Ulms, G., Hansen, K., Liu, J., Cow-drey, C., Yang, J., DeArmond, S. J., Cohen, F. E., Prusiner, S. B., and Baldwin, M. A. (2004) Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues. Nat Biotechnol 22, 724–31.
Vasilescu, J., Guo, X., and Kast, J. (2004) Identification of protein–protein interactions using in vivo cross-linking and mass spec-trometry. Proteomics 4, 3845–54.
Anjaneyulu, P. S., and Staros, J. V. (1987) Reactions of N -hydroxysulfosuccinimide active esters. Int J Pept Protein Res 30, 117–24.
Schulz, D. M., Ihling, C., Clore, G. M., and Sinz, A. (2004) Mapping the topology and determination of a low-resolution three-dimensional structure of the calmodulin–melittin complex by chemical cross-linking and high-resolution FTICRMS: Direct demonstration of multiple binding modes. Biochemistry 43, 4703–15.
Rappsilber, J., Siniossoglou, S., Hurt, E. C., and Mann, M. (2000) A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry. Anal Chem 72, 267–75.
Acknowledgments
This research was supported by the Office of Science (BER), US Department of Energy, Grant No. DE-FG02–04ER63924 and NIH-NCRR, Grant No. 1 R01 RR023334-01A1.
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Tang, X., Bruce, J.E. (2009). Chemical Cross-Linking for Protein–Protein Interaction Studies. In: Lipton, M.S., Paša-Tolic, L. (eds) Mass Spectrometry of Proteins and Peptides. Methods In Molecular Biology, vol 492. Humana Press. https://doi.org/10.1007/978-1-59745-493-3_17
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DOI: https://doi.org/10.1007/978-1-59745-493-3_17
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