Flash Oxidation (FOX) System: A Novel Laser-Free Fast Photochemical Oxidation Protein Footprinting Platform

J Am Soc Mass Spectrom. 2021 Jul 7;32(7):1601-1609. doi: 10.1021/jasms.0c00471. Epub 2021 Apr 19.


Hydroxyl radical protein footprinting (HRPF) is a powerful and flexible technique for probing changes in protein topography. With the development of the fast photochemical oxidation of proteins (FPOP), it became possible for researchers to perform HRPF in their laboratory on a very short time scale. While FPOP has grown significantly in popularity since its inception, adoption remains limited due to technical and safety issues involved in the operation of a hazardous Class IV UV laser and irreproducibility often caused by improper laser operation and/or differential radical scavenging by various sample components. Here, we present a new integrated FOX (Flash OXidation) Protein Footprinting System. This platform delivers sample via flow injection to a facile and safe-to-use high-pressure flash lamp with a flash duration of 10 μs fwhm. Integrated optics collect the radiant light and focus it into the lumen of a capillary flow cell. An inline radical dosimeter measures the hydroxyl radical dose delivered and allows for real-time compensation for differential radical scavenging. A programmable fraction collector collects and quenches only the sample that received the desired effective hydroxyl radical dose, diverting the carrier liquid and improperly oxidized sample to waste. We demonstrate the utility of the FOX Protein Footprinting System by determining the epitope of TNFα recognized by adalimumab. We successfully identify the surface of the protein that serves as the epitope for adalimumab, identifying four of the five regions previously noted by X-ray crystallography while seeing no changes in peptides not involved in the epitope interface. The FOX Protein Footprinting System allows for FPOP-like experiments with real-time dosimetry in a safe, compact, and integrated benchtop platform.

MeSH terms

  • Chromatography, Liquid
  • Epitopes / chemistry
  • Equipment Design
  • HEK293 Cells
  • Humans
  • Oxidation-Reduction
  • Protein Conformation
  • Protein Footprinting / instrumentation*
  • Protein Footprinting / methods*
  • Tandem Mass Spectrometry
  • Tumor Necrosis Factor-alpha / analysis
  • Tumor Necrosis Factor-alpha / chemistry
  • Tumor Necrosis Factor-alpha / genetics


  • Epitopes
  • Tumor Necrosis Factor-alpha