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Title: Characterizing Enzyme Cooperativity with Imaging SAMDI‐MS

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.

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Author(s) / Creator(s):
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Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Chemistry – A European Journal
Medium: X
Sponsoring Org:
National Science Foundation
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  1. Abstract

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    This article was corrected on 19 July 2022. See the end of the full text for details.

    Basic Protocol 1: Lentivirus production and expression line creation

    Support Protocol 1: Six‐well assay for estimation of production cell line yield

    Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His‐tags

    Basic Protocol 2: Cultures for production of Class II MHC proteins

    Basic Protocol 3: Purification of Class II MHC proteins by anti‐leucine zipper affinity chromatography

    Alternate Protocol 1: IMAC purification of His‐tagged Class II MHC

    Support Protocol 3: Protein concentration measurements and adjustments

    Support Protocol 4: Polishing purification by anion‐exchange chromatography

    Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation

    Basic Protocol 4: Peptide exchange

    Basic Protocol 5: Analysis of peptide exchange by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry

    Alternate Protocol 2: Native isoelectric focusing to validate MHC‐II peptide loading

    Basic Protocol 6: Multimerization

    Basic Protocol 7: Staining cells with Class II MHC tetramers

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