X-Ray Footprinting

Technique: X-ray Footprinting (also Synchrotron Footprinting)

Hydroxyl-radical-mediated (•OH) protein footprinting using mass spectrometry has recently been developed to define protein structure, assembly, and conformational changes in solution based on measurements of reactivity of amino acid side-chain. •OH radicals suitable for footprinting experiments can be generated by multiple methods such as Fenton reagent, from photo-oxidation of peroxide, using electrical discharge and from radiolysis of water. Synchrotron footprinting is a technology (SF) that combines a number of state-of-the-art techniques. Radiolysis of water using X-rays from synchrotron sources generates •OH radicals isotropically in solution without addition of chemicals. •OH radicals generated through millisecond exposures of synchrotron X-rays can then react with proteins to yield stable oxidative modifications of solvent-accessible amino acid side chains. Subsequent to oxidation, proteins are digested by specific proteases to generate peptides for mass spectrometry analysis. Accurate measurements of side-chain reactivity are achieved by quantitative liquid-chromatography-coupled MS. Also the oxidized residues can be identified using tandem MS. The reactivity of side chains with •OH can give insights into protein structure and monitor conformational changes (e.g. due to ligand binding or macromolecular interactions).

For more information on the X28C Beamline, click here.

Contact Person:
Sayan Gupta, Ph.D. (sayan@bnl.gov)
Brookhaven National Laboratory
National Synchrotron Light Source
Bldg. 725A-X28C
Upton, NY 11973
Phone: (631) 344-3725
Fax: (631) 344-5496