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This paper describes a method that combines a microfluidic device and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI) mass spectrometry to calculate the cooperativity in binding of calcium ions to peptidylarginine deiminase type 2 (PAD2). This example uses only 120 μL of enzyme solution and three fluidic inputs. This microfluidic device incorporates a self-assembled monolayer that is functionalized with a peptide substrate for PAD2. The enzyme and different concentrations of calcium ions are 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 Ca 2+ . 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. Analysis of the peptide conversion as a function of position and time gives the degree of cooperativity (n) and the concentration of ligand required for half maximal activity (K 0.5 ) for the Ca 2+ - 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., (© 2021 Wiley-VCH GmbH.)

Phosphorylation controls important cellular signals and its dysregulation leads to disease. While most phospho-regulation studies are focused on kinases, phosphatases are comparatively overlooked. Combining peptide arrays with SAMDI mass spectrometry, we show that tyrosine phosphatase activity is restricted by basic amino acids adjacent to phosphotyrosines. We validate this model using two β-catenin mutants associated with cancer (T653R/K) and a mouse model for intellectual disability (T653K). These mutants introduce a basic residue next to Y654, an established phosphorylation site where modification shifts β-catenin from cell–cell adhesions and towards its essential nuclear role as Wnt-signaling effector. We show that T653-basic mutant β-catenins are less efficiently dephosphorylated by phosphatases, leading to sustained Y654 phosphorylation and elevated Wnt signals, similar to those observed for Y654E phospho-mimic mutant mice. This model rationalizes how basic mutations proximal to phosphotyrosines can restrict counter-regulation by phosphatases, providing new mechanismistic and treatment insights for 6000+ potentially relevant cancer mutations. [ABSTRACT FROM AUTHOR]

This paper describes a method that combines a microfluidic device and self‐assembled monolayers for matrix‐assisted laser desorption/ionization mass spectrometry (SAMDI) mass spectrometry to calculate the cooperativity in binding of calcium ions to peptidylarginine deiminase type 2 (PAD2). This example uses only 120 μL of enzyme solution and three fluidic inputs. This microfluidic device incorporates a self‐assembled monolayer that is functionalized with a peptide substrate for PAD2. The enzyme and different concentrations of calcium ions are 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. Analysis of the peptide conversion as a function of position and time gives the degree of cooperativity (n) and the concentration of ligand required for half maximal activity (K0.5) 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. [ABSTRACT FROM AUTHOR]