Abstract
Mass spectrometric analyses of proteins affinity-purified from cell lysates are routinely used by cell biologists to characterize the composition and the modifications of protein complexes. Here, we describe a protocol that combines affinity-purification with chemical cross-linking and mass spectrometry (CXMS) in order to detect spatially proximate lysine residues on protein complexes isolated from human tissue culture cells. These cross-links are interpreted as distance restraints that aid in elucidating protein binding interfaces and the topology of protein complexes. In contrast to established high-resolution structural biology techniques, CXMS analysis has the potential to acquire structural information of small amounts of structurally flexible and heterogeneous protein preparations. We recently demonstrated on a network of modular protein phosphatase 2A complexes that restraints obtained by CXMS analysis hold great promise in supporting hybrid structural analysis of endogenous protein complexes by integrating structural data from different sources with computational molecular modeling.
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Acknowledgments
I would like to thank Alexander Leitner for comments on the manuscript and Thomas Walzthöni and Ruedi Aebersold for support. The work in the lab of F.H. is funded by the Bavarian Research Center for Molecular Biosystems and by an LMUexcellent Junior grant.
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Herzog, F. (2014). Measuring Spatial Restraints on Native Protein Complexes Using Isotope-Tagged Chemical Cross-Linking and Mass Spectrometry. In: Chen, Y. (eds) Structural Genomics. Methods in Molecular Biology, vol 1091. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-691-7_19
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DOI: https://doi.org/10.1007/978-1-62703-691-7_19
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