Periodic Reporting for period 2 - SynLink (Revealing the Synapse Architecture and Plasticity by Structural Interactomics)
Berichtszeitraum: 2022-07-01 bis 2023-12-31
Protein interactions play a central role brain function. Understanding protein-protein interactions (PPIs) during synaptic transmission is thus crucial in the discovery of new therapeutic interventions in neurological and psychiatric disorders. In the project, I proposed to develop new tools in an emerging technique, namely cross-linking mass spectrometry, to probe the entire protein interaction network at the synapse systematically. During the first funding period, our team has developed a novel membrane permeable and enrichable cross-linker tBu-PhoX to advance the depth and sensitivity of PPI detection. This allowed us to generate so far the largest protein interactome dataset from the synapse, which opens many doors for the discovery of important regulatory mechanisms in synapse function. Furthermore, we developed a quantitative cross-linking mass spectrometry pipeline, which allows us to effectively study interactome changes in the progression of neurological diseases.
In the first funding period, we have made substantial methodological advancements proposed in WP1 and WP2. For WP1, we developed a novel membrane permeable and IMAC-enrichable cross-linker tBu-PhoX, which gives an extremely satisfactory enrichment efficiency and specificity. Using this cross-linker, we achieved a five-fold higher cross-link identification number with substantially reduced analysis time in model systems. This work has been published in Angewandte Chemie in 2021 (Jiang P., Wang C., et al., A Membrane-Permeable and Immobilized Metal Affinity Chromatography (IMAC)-Enrichable Cross-Linking Reagent to Advance In Vivo Cross-Linking Mass Spectrometry. Angewandte Chemie, 2021). For WP2, we have established and optimized TMT-based quantitative cross-linking mass spectrometry (qXL-MS) pipeline and tested it on a benchmarking two-interactome system. We selected the qXL-MS method that balances identification sensitivity and quantification accuracy. We have thoroughly tested the pipeline and made it readily applicable to the synaptic system proposed in WP4. This work has been published in Anal Chem in 2022 (Ruwolt M., et al., Optimized TMT-based quantitative cross-linking mass spectrometry strategy for large-scale interactomic studies. Anal Chem. 2022).
Furthermore, using a commercially available non-enrichable cross-linker DSSO, we have generated by far the largest synaptic interactome dataset using the sequential digestion protocol proposed in WP3. We are in the progress of dissecting this extremely rich dataset systematically and looking for new candidates to follow up. Meanwhile, we will also repeat this protocol using our newly developed tBu-PhoX cross-linker, which will further improve the coverage and depth of the synaptic interactome proposed in WP3 and WP4.
Furthermore, using a commercially available non-enrichable cross-linker DSSO, we have generated by far the largest synaptic interactome dataset using the sequential digestion protocol proposed in WP3. We are in the progress of dissecting this extremely rich dataset systematically and looking for new candidates to follow up. Meanwhile, we will also repeat this protocol using our newly developed tBu-PhoX cross-linker, which will further improve the coverage and depth of the synaptic interactome proposed in WP3 and WP4.
I anticipate at the end of the project we will generate an unprecedented PPI network repository that are available in public. This information would be of great helpful for biologists in particular neuroscientists in dissecting the mechanistic functions of synapse at the molecular level. Meanwhile, I also aim to find new PPIs that are involved in important regulatory roles in synaptic function and I will also use complementary approaches to understand their functions and the biological consequences of their mis-regulation.