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J Am Chem Soc. 2006 Aug 16;128(32):10362-3.

Isotope-coded and affinity-tagged cross-linking (ICATXL): an efficient strategy to probe protein interaction surfaces.

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Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.


Chemical cross-linking followed by identification of the cross-linked residues by mass spectrometry provides structural information on protein interaction surfaces. Nevertheless, accurate analysis of the digested, cross-linked proteins is often challenging. Herein, we describe a novel strategy that relies on the use of affinity-tagged cross-linkers and isotope coding on the cross-linker-modified species. Incorporation of O16 or O18 during the hydrolysis of the cross-linkers results in a characteristic "doublet" for the undesired products of a half-cross-linking reaction. Therefore, genuine cross-linked peptides are readily distinguished for further structural analysis. This strategy permits a sensitive and facile analysis on a dimeric protease inhibitor, ecotin, showing general applicability to other protein assemblies.

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