Improving identification of in-organello protein-protein interactions using an affinity-enrichable, isotopically-coded, and mass spectrometry-cleavable chemical crosslinker

An experimental and computational approach for identification of protein-protein interactions by in-vivo chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically-coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on a proteome-wide scale. Both known and previously-unknown protein-protein interactions were able to be identified.