This dissertation is focused on developing cross-linking and mass spectrometry methodologies to study protein-protein interactions. Top-down cross-linking, in combination with mass spectrometry, provides advantages over bottom-up approaches, such as retaining posttranslational modification. Intermolecular cross-linking studies focus on defining protein complex topology and protein-protein interactions. We first developed the top-down MS approach to analyze intermolecular cross-linking in human hemoglobin. Both α-α and β-β intermolecular cross-linking were found and the cross-linking sites on the protein were identified, obtaining distance constraints between subunits of the human hemoglobin protein complex. This methodology would be a promising approach to characterize protein complexes and protein-protein interactions with high throughput and automation.
This dissertation also focuses on development of cross-linking mass spectrometry to study synphilin-1 interactors and aggresome formation. Synphilin-1 is a protein that promotes the formation of protein aggregates and aggresome formation upon proteasome inhibition, and is implicated in Parkinson disease. Synphilin-1 contains several protein binding motifs. The biological functions of synphilin-1 and its role in aggresome formation and the pathogenesis of Parkinson disease remain to be elucidated. We utilized tandem affinity purification and label-free mass spectrometry to explore the patterns of cellular proteins associated with synphilin-1. We identified 57 synphilin-1 interacting proteins, and functional enrichment and pathway analysis showed that many of the associated proteins are involved in chromatin modulation, RNA and protein metabolism. Furthermore, we developed a proteomic strategy to identify synphilin-1 binary interacting partners as well as interacting domains using affinity purification followed by isotopically tagged cross-linking in combination with mass spectrometry. We found 24 newly discovered proteins that directly bind to synphilin-1. The proteins were mainly involved in RNA metabolism. The coiled-coil domain (CC), ankyrin-like repeat domain 2 (ANK2), and the protein aggregate promoting domain, appeared to the main regions that bound proteins. The functions of synphilin-1 interacting proteins, such as CK2, in aggresome formation were studied. The results show that CK2 is an important regulator of aggresome formation, but not through its kinase activities. Involvement of synphilin-1 in autophagy was also investigated. Knockdown of synphilin-1 shows that synphilin-1 impacts autophagy.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/14097 |
| Date | 22 January 2016 |
| Creators | Xu, Xiaobin |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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