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

• Published in: Molecular & cellular proteomics 2020-04, Vol.19 (4), p.624-639
• Main Authors: Makepeace, Karl A.T., Mohammed, Yassene, Rudashevskaya, Elena L., Petrotchenko, Evgeniy V., Vögtle, F.-Nora, Meisinger, Chris, Sickmann, Albert, Borchers, Christoph H.
• Format: Article
• Language: English
• Subjects: Biotin - analogs & derivatives; Chemical Fractionation; Cross-Linking Reagents - chemistry; Crosslinking; Isotope Labeling; Mass Spectrometry; Mitochondria - metabolism; mitochondria function or biology; Models, Molecular; organelle-wide interactions; Organelles - metabolism; Peptides - metabolism; protein complex analysis; Protein Interaction Mapping - methods; Protein Interaction Maps; protein-protein interactions; Saccharomyces cerevisiae - metabolism; Succinimides
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.RA119.001839

By using an enrichable, 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. Our method applied to isolated yeast mitochondria allowed us to observe protein-protein interactions involving approximately one quarter of the proteins in the mitochondrial proteome. Our approach is suitable for proteome-wide applications, and facilitates investigations into condition-specific protein conformations, protein-protein interactions, system-wide protein function or dysfunction, and diseases. [Display omitted] Highlights •Used affinity-enrichable, isotopically coded, and MS-cleavable crosslinker.•Targeted acquisition strategy based on isotopic-coding described and evaluated.•Novel data analysis pipeline developed provides improved crosslink identification.•Large dataset reveals hundreds of mitochondrial protein-protein interactions. 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.