Improving Identification of In-organello Protein-Protein Interactions Using an Affinity-enrichable, Isotopically Coded, and Mass Spectrometry-cleavable Chemical Crosslinker

Date

2020

Authors

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

Journal Title

Journal ISSN

Volume Title

Publisher

Molecular & Cellular Proteomics

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.

Description

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Citation

Makepeace, K. A. T., Mohammed, Y., Rudashevskaya, E. L., Petrotchenko, E. V., Vögtle, F., Meisinger, C., … Borchers, C. H. (2020). Improving Identification of Inorganello Protein-Protein Interactions Using an Affinity-enrichable, Isotopically Coded, and Mass Spectrometry-cleavable Chemical Crosslinker. Molecular & Cellular Proteomics, 19(4), 624-639. https://doi.org/10.1074/mcp.RA119.001839.