Label-free duplex SAMDI-MS screen reveals novel SARS-CoV-2 3CLpro inhibitors.

The 3-chymotrypsin-like cysteine protease (3CLpro) of severe acute respiratory syndrome conoravirus 2 (SARS-CoV-2) remains a promising therapeutic target to combat COVID-19. Our group recently described a novel duplexed biochemical assay that combines self-assembled monolayers of alkanethiolates on gold with matrix assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS) to simultaneously measure 3CLpro and human rhinovirus 3C protease activities. This study describes applying the assay for the completion of a high-throughput duplexed screen of 300,000 diverse, drug-like small molecules in 3 days.

Exploring the Ligand Preferences of the PHD1 Domain of Histone Demethylase KDM5A Reveals Tolerance for Modifications of the Q5 Residue of Histone 3.

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 binding-induced enhancement in the activity of a reporter enzyme, in combination with fluorescence polarization.

A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1.

The enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the conversion of isocitrate to alpha-ketoglutarate (αKG) and has emerged as an important therapeutic target for glioblastoma multiforme (GBM). Current methods for assaying IDH1 remain poorly suited for high-throughput screening of IDH1 antagonists. This paper describes a high-throughput and quantitative assay for IDH1 that is based on the self-assembled monolayers for matrix-assisted laser desorption/ionization-mass spectrometry (SAMDI-MS) method.

High-throughput mapping of CoA metabolites by SAMDI-MS to optimize the cell-free biosynthesis of HMG-CoA.

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.

Using Microfluidics and Imaging SAMDI-MS To Characterize Reaction Kinetics.

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.

An Assay Based on SAMDI Mass Spectrometry for Profiling Protein Interaction Domains.

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.