Metabolic engineering uses enzymes to produce small molecules with industrial, pharmaceutical, and energy applications. However, efforts to optimize enzymatic pathways for commercial production are limited by the throughput of assays for quantifying metabolic intermediates and end products. We developed a multiplexed method for profiling CoA-dependent pathways that uses a cysteine-terminated peptide to covalently capture CoA-bound metabolites. Captured metabolites are then rapidly separated from the complex mixture by immobilization onto arrays of self-assembled monolayers and directly quantified by SAMDI mass spectrometry. We demonstrate the throughput of the assay by characterizing the cell-free synthesis of HMG-CoA, a key intermediate in the biosynthesis of isoprenoids, collecting over 10,000 individual spectra to map more than 800 unique reaction conditions. We anticipate that our rapid and robust analytical method will accelerate efforts to engineer metabolic pathways.

This paper describes an assay that can profile the binding of a protein to ligands and can rank the affinities of a library of ligands. The method is based on the enhanced rate of an enzyme-mediated reaction that follows from colocalization of the enzyme and substrate by a protein-ligand interaction. This assay uses a self-assembled monolayer that presents a candidate peptide ligand for a receptor and a peptide substrate for an enzyme. The receptor is prepared as a fusion to the relevant enzyme so that binding of the receptor to the immobilized ligand brings the enzyme to the surface, where it can more rapidly modify its substrate. The extent of conversion of the substrate to product is therefore a measure of the average time the ligand-receptor complex is present and is quantified using the SAMDI mass spectrometry technique. The approach is used to profile the binding of chromodomain proteins to methylated lysine peptides derived from the histone 3 protein. The relative affinities for the peptide ligands found in this work agreed with results from prior studies. Additionally, this work revealed cross-talk interactions whereby phosphorylation of certain residues impaired binding of chromodomains to the peptide ligands. The method presented here, which we term protein interaction by SAMDI (PI-SAMDI), has the advantages that it is applicable to low-affinity interactions because the complexes are not observed directly, but rather leave a "covalent record" of the interaction that is measured with mass spectrometry and because it is compatible with laboratory automation for high-throughput analysis.

This paper reports a SAMDI-mass spectrometry assay that can evaluate antagonists of cancer-associated isocitrate dehydrogenase 1 in high throughput.

Microfluidic platforms have enabled the simplification of biochemical assays with a significant reduction in the use of reagents, yet the current methods available for analyzing reaction products can limit applications of these approaches. This paper demonstrates a simple microfluidic device that incorporates a functionalized self-assembled monolayer to measure the rate constant for a chemical reaction. The device mixes the reactants and allows them to selectively immobilize to the monolayer at the base of a microfluidic channel in a time-dependent manner as they flow down the channel. Imaging self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (iSAMDI-MS) is used to acquire a quantitative image representing the time-resolved progress of the reaction as it flowed through the channel. Knowledge of the surface immobilization chemistry and the fluid front characteristics allows for the determination of the chemical reaction rate constant. This approach widens the applicability of microfluidics for chemical reaction monitoring and establishes a label-free method for studying processes that occur within a dispersive regime.

Understanding the ligand preferences of epigenetic reader domains enables identification of modification states of chromatin with which these domains associate and can yield insight into recruitment and catalysis of chromatin-acting complexes. However, thorough exploration of the ligand preferences of reader domains is hindered by the limitations of traditional protein–ligand binding assays. Here, we evaluate the binding preferences of the PHD1 domain of histone demethylase KDM5A using the protein interaction by SAMDI (PI-SAMDI) assay, which measures protein–ligand binding in a high-throughput and sensitive manner via binding-induced enhancement in the activity of a reporter enzyme, in combination with fluorescence polarization. The PI-SAMDI assay was validated by confirming its ability to accurately profile the relative binding affinity of a set of well-characterized histone 3 (H3) ligands of PHD1. The assay was then used to assess the affinity of PHD1 for 361 H3 mutant ligands, a select number of which were further characterized by fluorescence polarization. Together, these experiments revealed PHD1’s tolerance for H3Q5 mutations, including an unexpected tolerance for aromatic residues in this position. Motivated by this finding, we further demonstrate a high-affinity interaction between PHD1 and recently identified Q5-serotonylated H3. This work yields interesting insights into permissible PHD1-H3 interactions and demonstrates the value of interfacing PI-SAMDI and fluorescence polarization in investigations of protein–ligand binding.