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A systematic and validated Fe(II)-EDTA method of hydroxyl radical generation for oxidative protein footprinting

ABSTRACT: Correlating the structure and dynamics of proteins with biological function is critical to understanding normal and dysfunctional cellular mechanisms. We describe a quantitative method of hydroxyl radical generation via Fe(II)-EDTA catalyzed Fenton chemistry that provides easy access to oxidative protein footprinting using equipment commonly found in research and process control laboratories. Robust and reproducible dose-dependent oxidation of protein samples is observed and quantitated by mass spectrometry with as fine as single residue resolution. An oxidation analysis of lysozyme provides a readily accessible benchmark for our method. The efficacy of our oxidation method is demonstrated through mapping the interface of a RAS monobody complex, the surface of the NIST mAb, and the interface between PRC2 complex components. These studies are executed using standard laboratory tools and a few pennies of reagents; the mass spectrometry analysis can be streamlined to map protein structure with single amino acid residue resolution. All mass spectrometry raw files for this study are included here.

INSTRUMENT(S): Orbitrap Fusion Lumos, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Beatrix Ueberheide

PROVIDER: MSV000092853 | MassIVE |

SECONDARY ACCESSION(S): PXD045359

REPOSITORIES: MassIVE

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A systematic and validated Fe(II)-EDTA method of hydroxyl radical generation for oxidative protein footprinting

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