16598
A Microbead-Based Multiplex Immunoassay to Measure Dynamic Protein Interaciton Networks at the Glutamate Synapse
Objectives: To develop a technique to quantitatively measure protein-protein interactions at the network level, and to quantify how these networks change in response to biological signaling events and autism-linked gene mutations.
Methods: We have developed a technique called multiplex immunoprecipitation detected by flow cytometry (mIP-FCM). In mIP-FCM, protein complexes are immunoprecipitated onto Luminex microspheres conjugated to antibodies specific for 20+ different proteins, then probed with fluorophore-conjugated antibodies to the same protein set and read on a flow cytometer. In total, 400+ protein-protein interactions are assessed, producing a network-scale analysis of protein interactome dynamics. Our group uses mIP-FCM to study the signaling networks engaged in response to agonist vs. antagonist stimuli of either the T cell immunologic synapse or the neuronal glutamatergic synapse. Here, we present a new mIP-FCM array focused on autism-linked proteins from the glutamate synapse.
Results: Using the well-characterized T cell immunologic synapse that forms between a T cell and an antigen presenting cell as a model system, mIP-FCM is capable of assessing 400+ dynamic interactions during a T cell signaling event. We have developed high-sensitivity statistical methods capable of identifying small changes in protein interactions, and visualization methods to make sense of the vast amount of data collected (~7000+ data points per timepoint). Analysis of proteins from the T cell synapse has revealed new network protein interaction patterns that could be decisive in agonist vs. antagonist signal transduction. In our new mIP-FCM array focused on the glutamate synapse, we have targeted 25 autism-related proteins that form a highly interconnected protein interaction network. Our preliminary data focuses on detecting changes in the interaction network specific to excitation or inhibition of synaptic activity.
Conclusions: Our goal is to better understand the network-scale alterations in protein-protein interactions during glutamatergic signaling, and how different autism-associated risk factors may play important, perhaps convergent, roles in these synaptic network processes. The data presented here are an important first step towards this goal.