Development of Broad‐Based Labeling Reagents for Peptide and Protein Footprinting

Abstract

Interactions between macromolecules in living organisms are governed by fundamental biophysical principles. Different methods to study these biophysical properties exist; including footprinting. Footprinting refers to the structural probing of interaction between macromolecules using chemical reactions ranging from HDX to reactive species such as free radicals and carbenes. Modern footprinting methods allows for a fast, specific, and precise determination of proteins' structure, interaction sites, conformational changes, folding, misfolding, and binding affinity. The complete understanding of these, in‐vitro and in the cell, is crucial for drug development and antibody synthesis for therapeutics and diagnostics. These advances contribute to the understanding of the human proteome. Methods for determining a protein footprint include Mass Spectrometry (MS), which is advantageous given its sensitivity. Covalent modification of amino acid residues coupled with MS has been useful in characterizing non‐membrane and membrane proteins; a field where current biophysical techniques are limited. Covalent modification can be achieved via Fast Photochemical Oxidation of Proteins (FPOP) using radical chemistry. Residue labeling using current radical species lacks generality or broad reactivity among amino acids. Thus, the development of novel chemical labeling methods that increases amino acid labeling are needed. To implement this, we propose the use of carbene species (double radicals), trifluoro‐methyl radicals, and chloride cations. Carbene compounds will be generated via FPOP from diazirine precursors, which were synthesized from ketone precursors. The Togni's Reagent will be used as a precursor for covalent modification through trifluoromethyl radicals and Palau's reagent will be used for modification through chloride cations. If a robust footprinting method is developed to further understand protein biophysics it would lead to further advances in the study of protein folding, protein‐protein interactions, and drug development.Support or Funding InformationAmgen Foundation. Michael Gross's Laboratory. NIH/NIGMS Biomedical Mass Spectrometry Resource.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.