Initially, to improve protein identification by mass spectrometry I developed an approach to chemically modify streptavidin beads (protease-resistant streptavidin). In fact, purification of biomolecules via streptavidin is extensively utilized in molecular biology and biochemistry to identify biotinylated biomolecules and their interaction partners. However intense streptavidin-derived peptides affect protein identification by mass spectrometry. My approach for chemical modification of streptavidin reduces the contaminations over 100-fold. As a result, proteins interacting with diverse biotinylated bait-molecules such as DNA, proteins and lipids are identified with deeper coverage. The results were published in a peer-reviewed journal (Rafiee MR et al. MSB 2020).
After that I developed SPACE (Silica Particle Assisted Chromatin Enrichment), a straightforward and highly sensitive method for isolation of chromatin and identification of RBP using mass spectrometry. I evaluated SPACE by studying the global chromatin composition of mES cells. I successfully identified previously reported DNA- and chromatin-binding proteins, as well as many RBPs. Surprisingly, RBPs comprise ~48% of the proteins obtained from the chromatin composition. To understand how RBPs bind to chromatin, I developed SPACEmap which indicates that intrinsically disordered regions (IDRs) are frequently employed by chromatin proteins, including chromatin-associated RBPs, for chromatin-binding. I also took advantage of protease-resistant streptavidin to invent SPACE-SICAP, a more stringent version of SPACE which confirms previous findings with higher confidence. Altogether, I demonstrated various applications of SPACE and provided a systematic view of RBP-chromatin interactions. The results were submitted to a peer-reviewed journal, and is available in bioRxiv (
https://doi.org/10.1101/2020.07.13.200212(si apre in una nuova finestra))
I then harnessed the power of SPACE to interrogate chromatin proteome composition in neuronal differentiated from ALS-patient-induced pluripotent stem (iPS) cells. I find that mutant VCP has diminished chromatin-binding, which is associated with chromatin release of several DNA damage response factors, such as TP53BP1. Conversely, I observed increased chromatin recruitment of RNA quality control pathways in the mutant cells. These data highlight the critical function of VCP in promoting DNA repair such that ALS-causative VCP mutations make cells vulnerable to DNA damage. Currently, I am preparing a manuscript to publish the results.
