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
Keywords
UVic Genome BC Proteomics Centre
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.