Crosslinking and Mass Spectrometry for Identifying Protein-protein Interaction Sites in Activator-Multi-component Protein Complexes

摘要

The anaphase promoting complex (APC), a multi-component E3 ubiquitin ligase, is regulated by protein activators including Cdh1. Which APC subunit interacts with Cdh1 and the corresponding protein-protein interaction sites need to be identified. Here, we describe a strategy combining photochemical crosslinking and mass spectrometry to determine the specific preferential binding partner of Cdh1 and interaction sites. We use a modified Cdh1 C-terminal peptide containing photoactivatable benzophenone-phenylalanine and biotin groups. This photocrosslinking approach allows the capture of transient interaction between APC subunits and Cdh1 C-terminal peptide. The biotin group facilitates the detection of the peptide-interacting subunit by Western blot and the recovery of crosslinked peptides, which allows identification of the binding sites between Cdh1 C-terminal peptide and APC by mass spectrometry.