Chemical Generation of Hydroxyl Radical for Oxidative 'Footprinting'

Micheal Leser, Jessica R. Chapman, Michelle Khine, Jonathan Pegan, Matt Law, Mohammed El Makkaoui, Beatrix M. Ueberheide, Michael D. Brenowitz

Research output: Contribution to journalReview article

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.

Original languageEnglish (US)
Pages (from-to)61-69
Number of pages9
JournalProtein and peptide letters
Volume26
Issue number1
DOIs
StatePublished - Jan 1 2019

Fingerprint

Protein Footprinting
Hydroxyl Radical
Laminates
Proteins
DNA Footprinting
T-cells
Macromolecules
Gold
Nanoparticles
Nanocrystals
Nucleic Acids
Plastics
Mass spectrometry
Mass Spectrometry
Iron
RNA
Ligands
T-Lymphocytes
Equipment and Supplies
Oxidation

Keywords

  • Fenton reaction
  • footprinting
  • hydroxyl radical
  • mass spectrometry
  • protein
  • structure mapping.

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry

Cite this

Chemical Generation of Hydroxyl Radical for Oxidative 'Footprinting'. / Leser, Micheal; Chapman, Jessica R.; Khine, Michelle; Pegan, Jonathan; Law, Matt; Makkaoui, Mohammed El; Ueberheide, Beatrix M.; Brenowitz, Michael D.

In: Protein and peptide letters, Vol. 26, No. 1, 01.01.2019, p. 61-69.

Research output: Contribution to journalReview article

Leser, M, Chapman, JR, Khine, M, Pegan, J, Law, M, Makkaoui, ME, Ueberheide, BM & Brenowitz, MD 2019, 'Chemical Generation of Hydroxyl Radical for Oxidative 'Footprinting'' Protein and peptide letters, vol. 26, no. 1, pp. 61-69. https://doi.org/10.2174/0929866526666181212164812
Leser, Micheal ; Chapman, Jessica R. ; Khine, Michelle ; Pegan, Jonathan ; Law, Matt ; Makkaoui, Mohammed El ; Ueberheide, Beatrix M. ; Brenowitz, Michael D. / Chemical Generation of Hydroxyl Radical for Oxidative 'Footprinting'. In: Protein and peptide letters. 2019 ; Vol. 26, No. 1. pp. 61-69.
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