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.
