Development of Novel Technologies for Interactome Detection
Interactome refers to physical interactions among molecules in a particular cell. Interactomics is the fundamental study for revealing many types of biological phenomena. The components of interctome include protein-protein interactions, protein-ligand interactions, protein-DNA interactions and etc. In this dissertation, we focused on protein-protein interactions and protein-ligand interactions. We discussed the background and existing detection methods in Chapter 1, and presented our novel high-throughput methods to detect these two types of interactomes in Chapter 2, 3 and 4. In Chapter 2, we describe a novel method which couples Biomolecular Fluorescence Complementation (BiFC) Assays with Mass Spectrometry (MS) to identify protein-protein interactions, termed fluorescence complementation mass spectrometry (FCMS). With FCMS, weak and transient protein interactions were detected by mass spectrometry after being stabilized by association of fluorescent protein fragments. This method was specifically applied to kinase-substrate interactions, which are usually transient and difficult to capture and identify by traditional affinity purification-mass spectrometry (AP-MS) methods. Besides the identification of interactions, MS can also reveal Post Translational Modifications (PTMs), such as phosphorylation. To examine the feasibility of FCMS, we first tested it with Mitogen-activated protein kinase 3 (ERK1/MAPK3) and its substrates: cyclic AMP-dependent transcription factor ATF2, proto-oncogene c-Fos (Fos) and transcription factor AP-1 (Jun). Our experiments confirmed that FCMS is able to stabilize and detect the interactions between ERK1 and its substrates. Before expanding FCMS to a high-throughput method to study the upstream kinases of a substrate, the interaction between cAMP response element-binding protein (CREB) and its known kinases such as protein kinase A (PKA), casein kinase II (CKII), and glycogen synthase kinase 3 beta (GSK3b) was examined by FCMS. Our results indicate that FCMS is sensitive enough for high-throughput studies. In Chapter 3, FCMS was applied to explore the upstream kinases of CREB. Wild type or interaction resistant CREB mutants and a human kinase library were co-expressed in stable isotope labeling using amino acids in cell culture (SILAC) cells. Through identification and quantification of interacting kinases between two types of CREB, we obtained information on CREB interacting kinases. Then potential novel CREB kinases were validated by in vitro kinase assay with 32p ATP auto-radioactive detection. The phosphorylation sites were also identified by mass spectrometry and further confirmed with phosphorylation resistant CREB mutants. We successfully identified 6 known CREB kinases and validated 5 new CREB kinases. To the best of our knowledge, this is the first MS based high-throughput study that identifies direct upstream kinases. In Chapter 4, we describe a novel method to identify protein-ligand interactions. In this method, the protein stability change induced by ligand-binding was measured by pulse-proteolysis coupled with quantitative mass spectrometry. The filter-aided sample preparation (FASP) technique was used to filter out pulse-digested peptides, and the remaining proteins are digested by trypsin. Tandem mass tags (TMT) are applied to label and quantify resulting peptides, which reveals the ligand binding status. Using nicotinamide adenine dinucleotide (NAD) as the ligand, we successfully identified several known NAD binding proteins by the pulse-proteolysis-quantitative mass spectrometry method. The experimental data was examined bioinformatically by applying the computer assisted docking prediction method PatchSurfer developed by Dr. Kihara’s group. By these means we successfully identified several proteins as putative NAD-binding proteins with high confidence.
Tao, Purdue University.
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