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

Abstract

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.

Hydroxyl Radical Protein Footprinting: A Mass Spectrometry-Based Structural Method for Studying the Higher Order Structure of Proteins

“…Streamlining the FPOP workflow would further facilitate the adoption of these methods to a broad audience. Recently, a flash oxidation (FOX) system was developed for performing HRPF using a high-pressure flash lamp for photolysis of H 2 O 2 . This system eliminates the use of the laser, whose safety issues may limit the adoption of laser-based HRPF by some laboratories.…”

“…A comparison of Sequest and Bolt for IC-FPOP showed that Bolt identified a 1.5-fold greater number of modified peptides in 64 h of computational time compared to 203 h for Sequest, thus increasing throughput and the amount of structural information. Finally, the recently developed FoxWare protein footprinting software has been successful in quantification of HRPF modifications . Even with these advancements, data analysis is still the most time-consuming and complex part of HRPF studies.…”

Mass Spectrometry Methods for Measuring Protein Stability

“…FPOP experiments often require laser irradiation within flow injection manifolds to control the oxidation reactions used for protein labeling . More recent versions of FPOP operate without the need of lasers, offering the potential to measure protein stabilities directly through the direct observation of protein oxidation rates and are moving toward in vivo proteome-wide assessments of protein stability values. Future advancements in FPOP will likely continue along this trajectory, further enabling the measurement of protein and protein complex stabilities on a proteome scale. , …”

Multiplex chemical labeling of amino acids for protein footprinting structure assessment

“…10 Many methods of generating hydroxyl radicals have been developed over recent decades and are currently routinely employed, including radiolysis of water by X-rays, gamma rays, electron beams, electric discharge or a plasma source, decomposition of hydrogen peroxide using transition metal based Fenton chemistry and photolysis of hydrogen peroxide using lasers or a high-pressure flash oxidation lamp. [11][12][13][14][15] Independent of the method of production, •OH are highly reactive species and have van der Waals surface area and solvent properties similar to those of water molecules. 14 •OH are polar moieties, react irreversibly with side chains on the order of microseconds and can routinely modify 12 out of the 20 amino acids in HRPF experiments.…”