(595e) Characterizing Enzyme Cooperativity with Imaging Samdi-MS

Cooperative binding interactions are important in metabolic regulation, cellular signaling, and disease progression. Binding of a metal or ligand to multiple sites in a protein is described by the Hill coefficient (n) which describes the degree of cooperativity and where K_0.5describes the ligand concentration required for half maximal activity. However, there remains a need for high-throughput strategies to quantitatively measure cooperative binding interactions. Our group has have previously shown that we can overcome this challenge by using iSAMDI-MS (imaging self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry), which can perform and analyze thousands of reactions in a single experiment for calculating the Michaelis constant and rate of a chemical reaction. This talk describes the utility of iSAMDI for calculating the cooperativity in binding of calcium ions to peptidylarginine deiminase type 2 (PAD2). Here, we will evaluate the design elements for our microfluidic device, focusing on the incorporation of a self-assembled monolayer that is functionalized with a peptide substrate for PAD2. The enzyme and different concentrations of calcium ions were flowed through each of eight channels, where the position along the channel corresponds to reaction time and position across the channel corresponds to the concentration of Ca²⁺. Imaging SAMDI (iSAMDI) is then used to determine the yield for the enzyme reaction at each 200 μm pixel on the monolayer, providing a time course for the reactions. After extracting the imaging mass spectrometry data, we utilized numerical calculations to quantify the spatiotemporal conversion of the peptide substrate into the citrullinated product as a function of concentration, position, and flow rate. Ultimately, analysis of the peptide conversion as a function of position/time gives the degree of cooperativity (n) and the concentration of ligand required for half maximal activity (K_0.5) for the Ca²⁺ – dependent activation of PAD2. This work establishes a high-throughput and label-free method for studying enzyme-ligand binding interactions and widens the applicability of microfluidics and matrix-assisted laser desorption/ionization mass spectrometry (MALDI) imaging mass spectrometry.