Chemical cross‐linking and mass spectrometry to map three‐dimensional protein structures and protein–protein interactions

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Abstract
Closely related to studying the function of a protein is the analysis of its three‐dimensional structure and the identification of interaction sites with its binding partners. An alternative approach to the high‐resolution methods for three‐dimensional protein structure analysis, such as X‐ray crystallography and NMR spectroscopy, consists of covalently connecting two functional groups of the protein(s) under investigation. The location of the created cross‐links imposes a distance constraint on the location of the respective side chains and allows one to draw conclusions on the three‐dimensional structure of the protein or a protein complex. Recently, chemical cross‐linking of proteins has been combined with a mass spectrometric analysis of the created cross‐linked products. This review article describes the most popular cross‐linking reagents for protein structure analysis and gives an overview of the different available strategies that employ chemical cross‐linking and different mass spectrometric techniques. The challenges for mass spectrometry caused by the enormous complexity of the cross‐linking reaction mixtures are emphasized. The various approaches described in the literature to facilitate the mass spectrometric detection of cross‐linked products as well as computer software for data analyses are reviewed. © 2006 Wiley Periodicals, Inc., Mass Spec Rev 25:663–682, 2006