Improving Identification of In-organello Protein-Protein Interactions Using an Affinity-enrichable, Isotopically Coded, and Mass Spectrometry-cleavable Chemical Crosslinker.
Mol Cell Proteomics
; 19(4): 624-639, 2020 04.
Article
in En
| MEDLINE
| ID: mdl-32051233
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.
Key words
Full text:
1
Database:
MEDLINE
Main subject:
Mass Spectrometry
/
Organelles
/
Cross-Linking Reagents
/
Protein Interaction Mapping
/
Isotope Labeling
Type of study:
Diagnostic_studies
/
Prognostic_studies
Language:
En
Journal:
Mol Cell Proteomics
Journal subject:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Year:
2020
Type:
Article
Affiliation country:
Canada