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Chemical Generation of Hydroxyl Radical for Oxidative ‘Footprinting’

Author(s):

Michael Brenowitz *, Jessica R. Chapman , Beatrix Ueberheide , Michael Khine , Jonathan Pegan , Matt Law , Mohammed El Makkaoui and Micheal Leser   Pages 1 - 11 ( 11 )

Abstract:


Background: For almost four decades, hydroxyl radical chemically generated by Fenton chemistry has been a mainstay for the oxidative ‘footprinting’ of macromolecules.

Objective: In this article, we start by reviewing the application of chemical generation of hydroxyl radical to the development of oxidative footprinting of DNA and RNA and the subsequent application of the method to oxidative footprinting of proteins. We next discuss a novel strategy for generating hydroxyl radicals by Fenton chemistry that immobilizes catalytic iron on a solid surface (Pyrite Shrink Wrap laminate) for the application of nucleic acid and protein footprinting.

Method: Pyrite Shrink-Wrap Laminate is fabricated by depositing pyrite (Fe-S2, aka ‘fool’s gold’) nanocrystals onto thermolabile plastic (Shrinky Dink). The laminate can be thermoformed into a microtiter plate format into which samples are deposited for oxidation.

Results: We demonstrate the utility of the Pyrite Shrink-Wrap Laminate for the chemical generation of hydroxyl radicals by mapping the surface of the T-cell co-stimulatory protein Programmed Death – 1 (PD-1) and the interface of the complex with its ligand PD-L1.

Conclusion: We have developed and validated an affordable and reliable benchtop method of hydroxyl radical generation that will broaden the application of protein oxidative footprinting. Due to the minimal equipment required to implement this method, it should be easily adaptable by many laboratories with access to mass spectrometry.

Keywords:

structural mass spectrometry, protein footprinting, biotherapeutics, higher order structure, hydroxyl radical footprinting, covalent labeling

Affiliation:

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY , Proteomics Laboratory, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY , Proteomics Laboratory, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY , Departments of Biomedical Engineering, University of California, Irvine, CA , Departments of Biomedical Engineering, University of California, Irvine, CA , Departments of Chemistry, University of California, Irvine, CA , Departments of Chemistry, University of California, Irvine, CA , Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY



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