Traceless Immobilization of Analytes for High-Throughput Experiments with SAMDI Mass Spectrometry

Abstract

Label-free assays, and particularly those based on the combination of mass spectroscopy with surface chemistries, enable high-throughput experiments of a broad range of reactions. However, these methods can still require the incorporation of functional groups that allow immobilization of reactants and products to surfaces prior to analysis. In this paper, we report a traceless method for attaching molecules to a self-assembled monolayer for matrix-assisted laser desorption and ionization (SAMDI) mass spectrometry. This method uses monolayers that are functionalized with a 3-trifluoromethyl-3-phenyl-diazirine group that liberates nitrogen when irradiated and gives a carbene that inserts into a wide range of bonds to covalently immobilize molecules. Analysis of the monolayer with SAMDI then reveals peaks for each of the adducts formed from molecules in the sample. This method is applied to characterize a P450 drug metabolizing enzyme and to monitor a Suzuki-Miyaura coupling chemical reaction and is important because modification of the substrates with a functional group would alter their activities. This method will be important for high-throughput experiments in many areas, including reaction discovery and optimization.

Using Microfluidics and Imaging SAMDI-MS To Characterize Reaction Kinetics

“…9,22−27 We have also recently described a traceless immobilization method that uses a photogenerated carbene to rapidly and covalently immobilize a broad range of molecules, and this may serve as a more generalizable immobilization strategy in future applications. 28 We note that it will be important to verify that both the reactant and product undergo immobilization to the monolayer with the same rate constant—or to determine rate constants for each molecule—so that the immobilized fraction can be related to relative concentrations in solution. Hence, we anticipate many ways in which the methodology described here can be applied to other reactions.…”

High-throughput photocapture approach for reaction discovery

“…2A). This method, termed traceless immobilization SAMDI-MS (TI-SAMDI-MS), has been applied as a tool to characterize a P450 drug-metabolizing enzyme and to monitor a Suzuki-Miyaura reaction (28); however, this method has never been applied to high-throughput analysis (HTA) until this work.…”

A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

“…Larger enzyme substrates,when similarly functionalized with ac lickable functional group,w ould be expected to yield probes more representative of their archetype.T of urther improve hit identification, PECANc ould be used in conjunction with P450 fingerprinting, in which the activities of enzyme variants on multiple probes are correlated to their activities on the substrate of interest measured for asubset of the library. [40][41][42] Alternatively,l abel-free technologies,e nabling the tagging of unfunctionalized biomolecules [43,44] promise to avoid this bias altogether.…”