Time-resolved hydroxyl radical protein footprinting (HRPF) employing mass spectrometry analysis was originated and developed in the late 1990s in synchrotron radiolysis studies.
[9] Years later in 2005, researchers Hambly and Gross introduced a method for protein oxidation on the microsecond timescale using laser flash photolysis of hydrogen peroxide to generate hydroxyl radicals.
The rate or level of oxidation at the reactive amino acid side chains (Met, Cys, Trp, Tyr, Phe, His, Pro and Leu) provides a measure of their accessibility to the bulk solvent.
A further requirement is to generate hydroxyl radicals from the bulk solvent (i.e. water) (equations 1 and 2) not hydrogen peroxide which can remain to oxidize proteins even without other stimuli.
[18] The application of ion mobility mass spectrometry has conclusively demonstrated that the conditions employed in RP-MS/Protein footprinting experiments do not alter the structure of proteins.