Advances in Structural Biology Methods: Protein Footprinting with Radical Probe Mass Spectrometry and Complementary Techniques

Dear Colleagues,

Protein Footprinting coupled with mass spectrometry is a pioneering method in structural biology that enables the study of three-dimensional protein structures, folding and macromolecular inetractions dynamics on fast timescales (down to sub-milliseconds).  The methodology is based on hydroxyl radicals (•OH) generated directly from water within aqueous solutions, which react with solvent accessible amino acid side chains, inducing covalent modifications that are analyzed by mass spectrometry techniques. It was first introduced in 1998 at the American Society for Mass Spectrometry (ASMS) annual conference, with subsequent articles in 1999 describing the production of hydroxy radicals by “Synchrotron X-Ray Radiolysis” and“Electrospray Assisted Discharge” techniques. The first review article in 2001 highlighted its utility to probe protein structures and interactions by mass spectrometry, with subsequent reviews describing applications in chemical biology and structural proteomics.  A recent 2019 review article highlights the significant growth of “Protein Footprinting” over 20 years, and its applications resulting in over 200 publications to date.

The “Footprinting” terminology was coined in 1988 by Lendon Payne in reference to limited proteolysis methods for mapping protein antigen-monoclonal antibody contact sites. This terminology was adopted by Thomas Tulius in 1989 and later in 2001 by David Galas in reference to the protection from hydroxyl radical cleavage by a protein bound to DNA within a DNA-protein complex, which implies the protein making a footprint at a particular point of interaction.

The “Footprinting” terminology is most popular, although the initial 1999 terminoglogy of Radical Probe Mass Spectrometry (RPMS) is most suitable, which universally defines all approaches that utilize radicals to probe the structures of biomolecules, regardless of the method of radical production. These include synchrotron X-ray radiolysis, commonly referred to as Hydroxyl Radical Protein Footprinting (HRPF) or Xray Footprinting with Mass Spectrometry (XF-MS), electrical discharge, ozonolysis, laser photolysis including the Fast Photochemical Oxidation of Proteins (FPOP).

This special issue initiative invites articles with an overview of this fast growing technology, covering advances of hydroxyl radical production, updates in capabilities of Synchrotron Facilities, and a range of biological and pharmaceutical applications of protein footprinting.

Another aim of this special issue to focus on complementary techniques that have revolutionalised the study of protein structures and dynamics, such as “hydrogen-deuterium exchange” combined with NMR spectroscopy or mass spectrometry. This revolutionary technique has made possible to visualise kinetics of protein folding at near amino acid resolution. Tryptophan fluorescence spectroscopy is another important technique that has enabled capturing local structural changes and dynamics of proteins on fast time scales (e.g. picoseconds). X-ray crystallography is another invaluable tool in structural biology, which continues to contribute greatly to protein strucutre determination.

In addition to the application and development of the various techniques, this special issue also invites articles on historical perspectives and emerging techniques applied to the study of protein strucutre and dynamics.

Dr. Simin D. Maleknia

Guest Editor

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