Site-specific interactome profiling with chemically synthetic phosphopeptides

Abstract

Signal transduction is the process for cell to sense extracellular signals and generate cellular responses through series of molecular events. Biochemically, signal transduction relies on transient and dynamic protein-protein interactions (PPIs), which are often regulated by protein post-translational modifications (PTMs). Membrane receptors mediated the transformation of extracellular signals to intracellular signals by recognizing their extracellular ligands and phosphorylation of their cytoplasmic domains, which induced the formation of phosphorylation-dependent protein complexes. The adaptor proteins scaffolded the complex by providing both PTM-recognition domains and phosphorylation sites for the docking of additional subunits to enlarge the complex.

Affinity purification-mass spectrometry (AP-MS) remain the most widely applied technique for PPI profiling. Standard AP-MS analysis requires a large amount of starting material, which greatly limits the application of AP-MS on primary cells and clinical samples. We developed a streamlined, sensitive, and easily performed sample preparation method, termed fully integrated spin tip-based AP-MS technique (FISAP). It significantly reduced the sample amount and process time for potential large scale applications (Chapter 2). Next, to study site-specific phosphotyrosine (pTyr) dependent interactome for protein domains longer than 50 AA, we employed native chemical ligation (NCL) to overcome the limitation of traditional peptide synthesis. We applied this strategy and synthesized the central linear region between the PTB and SH2 domains of the adaptor protein Shc1 with site-specific phosphorylation. These modified peptides were employed for FISAP to investigate the site-specific interactions of Shc1(Chapter 3). Finally, we employed both peptide ligations and FISAP to study the pTyr-dependent interactome of 9 immune co-inhibitory receptors, which are important targets for current tumor immunotherapy (Chapter 4). In summary, we believe the FISAP and NCL-based protein fragment synthesis will be generally applicable for the study of dynamic PTM-dependent protein complexes, which is critical for biomedical applications.