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

Karl A.T. Makepeace, Yassene Mohammed, Elena L. Rudashevskaya, Evgeniy V. Petrotchenko, F. Nora Vögtle, Chris Meisinger, Albert Sickmann, Christoph H. Borchers

    Research output: Contribution to journalArticlepeer-review

    19 Citations (Scopus)

    Abstract

    An experimental and computational approach for identification of protein-protein interactions by ex 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 an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain.

    Original languageEnglish
    Pages (from-to)624-639
    Number of pages16
    JournalMolecular and Cellular Proteomics
    Volume19
    Issue number4
    DOIs
    Publication statusPublished - 2020

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