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.
Highlights
Graphical AbstractIsotopically labeled, mass spectrometry (MS)-cleavable crosslinking reagent, a targeted MS2 acquisition strategy, and a novel software pipeline tailored to integrating crosslinker-specific mass spectral information we improved the detection, acquisition, and identification of crosslinker-modified peptides
Author’s Choice los Improving Identification of In-organello Protein-Protein Interactions Using an Affinityenrichable, Isotopically Coded, and Mass Spectrometry-cleavable Chemical Crosslinker□S
Chemical crosslinking combined with mass spectrometry is a valuable method for attaining structural information about proteins and identifying protein-protein interactions
Summary
Isotopically labeled, MS-cleavable crosslinking reagent, a targeted MS2 acquisition strategy, and a novel software pipeline tailored to integrating crosslinker-specific mass spectral information we improved the detection, acquisition, and identification of crosslinker-modified peptides. 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. We have uncovered system-wide interaction patterns that would not be accessible through classic proteinchemistry research techniques
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