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

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Karl Makepeace

LAB HEAD: Christoph H. Borchers

PROVIDER: PXD014055 | Pride | 2020-03-17

REPOSITORIES: Pride

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

Project description: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.

2020-03-17 | PXD017066 | Pride

DNA-seq of mitochondrial DNA from mouse heart that is both heart SOD2 knockout and deficient for mitochondrial DNA repair

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