Protein Interaction Studies by Chemical Cross-Linking Using Isotope-Labeled and Trifunctional Cross-Linkers Combined with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

摘要

Chemical cross-linking of protein complexes combined with a mass spectrometric analysis of the reaction products allows a rapid mapping of protein interfaces, which is crucial for understanding protein/protein interactions [1,2]. The identification of cross-linked products from the complicated mixtures created after the cross-linking reaction, however, remains a daunting task. In order to facilitate the identification of cross-linked products, we explore the use of isotope-labeled cross-linkers, which are employed as 1:1 mixtures of non-deuterated and four times-deuterated derivatives, as well as trifunctional cross-linkers containing an additional biotin moiety. This allows a rapid identification of cross-linked products based on their characteristic isotope patterns or, alternatively, cross-linker-containing species are enriched by affinity purification on avidin beads.