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Updated: Sep 9, 2025

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation
Published on: June 21, 2021
A biophysical approach to studying N-terminal cysteine oxidase substrate preferences
Karishma Patel1, Yannasittha Jiramongkol2, Mark D White3
1School of Chemistry, The University of Sydney, Sydney, NSW, Australia; School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, Australia.
Abstract:
N-terminal cysteine oxidases (NCOs) regulate adaptive responses to hypoxia in animals and plants, functioning as enzymatic oxygen sensors which control the oxygen-dependant stability of proteins bearing an N-terminal cysteine residue (Nt-cys) through the N-degron pathway. However, the precise properties governing NCO activity and substrate selectivity remain poorly understood. This chapter details optimised protocols for investigating the binding interactions between 2-aminoethanethiol (cysteamine) dioxygenase (ADO), a mammalian NCO, and its Nt-cys substrates using surface plasmon resonance (SPR) spectroscopy. SPR, with its real-time monitoring capabilities and continuous-flow microfluidics systems, facilitates the detection of rapid and transient binding events, providing a robust and versatile platform for investigating enzyme-substrate and enzyme-ligand interactions. This biophysical approach offers a valuable tool for dissecting the molecular mechanisms underlying NCO substrate recognition and regulation, which may be broadly applicable to other enzyme systems and post-translational modification processes.
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