Fast Protein Footprinting by X-ray Mediated Radical Trifluoromethylation

Abstract

Synchrotron radiolysis generates hydroxyl radicals (^•OH) that are successful footprinting reagents. Here, we describe a new reagent for the synchrotron platform, the trifluoromethyl radical (^•CF₃). The radical is produced by ^•OH displacement of ^•CF₃ from sodium triflinate (Langlois reagent). Upon X-ray beam exposure, the reagent labels proteins extensively without any additional chemicals on a millisecond or shorter time scale. The ^•CF₃ is comparably reactive to ^•OH and produces footprinting information that complements that of ^•OH alone. This reagent in combination with ^•OH should enable novel chemistry for protein footprinting on the synchrotron platform.

Figure 1.

Proposed pathway for X-ray mediated radical trifluoromethylation for protein footprinting.

Figure 2.

(a) Deconvoluted mass spectra. CF₃-modified holo-Mb (front) and apo-Mb (middle) at 25 ms of X-ray irradiation. Apo-Mb (back) without x-ray irradiation shows no CF₃-modifications. (b) Stars represent number of trifluoromethylations (+68 Da), and circles represent H substitution by •OH. This figure adapted from Figure S1. Comparison of chemical reactivities for •OH and •CF₃ provided by a Venn diagram comparing residues reactive with •OH (in orange) and with •CF₃ (in light green); residues in the overlap region are reactive with both •OH and •CF_3.

Figure 3.

(a) EIC for peptide 119–133 from a 75 ms exposure of holo-Mb for CF₃ footprinting on the synchrotron platform. (b) EIC for peptide 64–77 taken from the 50 ms exposure of holo-Mb. EICs for the unmodified (blue), hydroxyl-radical modified (orange), CF₃-radical labeled (red), and di-modified (green) peptides confirmed via MS/MS are shown.

Figure 4.

Comparison of X-ray-induced •CF₃ and •OH at the AA residue level. Changes in the modification rates respond to heme binding, for the rates of •CF₃ modification (a) and of •OH modification (c). Changes mapped on to the structure of holo-Mb (PDB: 1WLA) to identify regions with increased (red), decreased (blue), or unchanged (white) rates of modification upon heme binding, for •CF₃ modification (b) and •OH modification (d).

Figure 5.

(a) The fraction of unmodified peptide versus exposure time at peptide 80–96. (b)The EIC of peptide 80–96.