Protein Footprinting

Protein footprinting involves the study of the surface of proteins to investigate protein structure or how they assemble and interact within a larger macromolecular assembly. This has been traditionally achieved through the treatment of proteins with hydroxyl radicals.

Time-resolved hydroxyl radical protein footprinting employing mass spectrometry analysis was developed over a decade ago in synchrotron radiolysis studies conduced by Maleknia et al. The same year, these authors reported on the use of an electrical discharge source to effect the oxidation of proteins on millisecond timescales as proteins pass from the electrosprayed solution into the mass spectrometer. These approaches have since been successfully applied in the analysis of protein structures, protein folding, protein dynamics, and protein–protein interactions.

Unlike nucleic acid, proteins were found to oxidize rather than cleave on these timescales. Analysis of the products by mass spectrometry reveals that proteins to are oxidized in a limited manner (some 10–30% of total protein) at a number of amino acid side chains across the proteins. 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. The mechanisms of side chain oxidation was explored by performing the radiolysis reactions in 18O-labeled water.

A critical feature of these experiments is the need to expose proteins to hydroxyl radicals for limited timescales on the order of 1–50 ms inducing 10-30% oxidation of total protein. 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.

  • Equation 1: H2O → H2O+• + e• + H2O*
  • Equation 2: H2O+• + H2O → H3O+ + HO•

Hydroxyl radicals can be produced in solution by an electrical discharge within a conventional atmospheric pressure electrospray ionization (ESI) source. When a high voltage difference (~8 keV) is held between an electrospray needle and a sampling orifice to the mass analyzer, radicals can be produced in solution at the electrospray needle tip. This method was the first employed to apply protein footprinting to the study of a protein complex. A computer program has also been written to help model protein complexes using data from the RP-MS/Protein footprinting approach. RP-MS/Protein footprinting studies of protein complexes can also employ computational approaches to assist with this modeling.

Read more about Protein FootprintingApplications, Method

Other articles related to "protein footprinting, proteins, protein":

Protein Footprinting - Method
... The exposure of proteins to a "white" X-ray beam of synchrotron light or an electrical discharge for tens of milliseconds provides sufficient oxidative modification to the ... By adjusting the time for radiolysis or which protein ions spend in the discharge source, a time-resolved approach is possible which is valuable for the study of protein dynamics ...

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