Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins

Weinberger, Scot R.; Chea, Emily E.; Sharp, Joshua S.; Misra, Sandeep K.

doi:10.3791/61861

Cited by 8 publications

(21 citation statements)

References 33 publications

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“…The custom high-pressure Xe flash lamp provides broad emission lines, with a high UV intensity at short wavelengths where hydrogen peroxide absorbs photons strongly. 18 A flash lamp electrode design coupled with a custom power supply allows for very short light pulses (10 μs fwhm) combined with a stable shot-to-shot light output at a set drive voltage (typical lamp t 1/2 ≈ 171 000 pulses by adenine dosimetry, Figure 2). The duration of the illumination is significantly longer than the lifetime of the hydroxyl radical under most FPOP conditions, 9,19 making the duration of the pulse the rate-limiting step on labeling lifetime.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…While these light outputs are not as brief as those found with KrF excimer lasers (∼20 ns), they are far shorter than typical exposures used for X-ray synchrotron radiolysis HRPF studies 3 and should be sufficient for thorough labeling of most proteins without oxidation-induced denaturation artifacts in the HRPF data. 18 FOX System Oxidation of a Model Peptide. The oxidation of a model peptide, angiotensin II (DRVYIHPF), was carried out using an early prototype version of the FOX photolysis module to determine if the profiles of peptide oxidation products generated were typical of other HRPF methods.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Fast and stable illumination is key for the FOX system to properly emulate laser-based FPOP experiments. The custom high-pressure Xe flash lamp provides broad emission lines, with a high UV intensity at short wavelengths where hydrogen peroxide absorbs photons strongly . A flash lamp electrode design coupled with a custom power supply allows for very short light pulses (10 μs fwhm) combined with a stable shot-to-shot light output at a set drive voltage (typical lamp t 1/2 ≈ 171 000 pulses by adenine dosimetry, Figure ).…”

Section: Resultsmentioning

confidence: 99%

“…Previously published work has shown that the FOX system compares favorably to the traditional laser-based FPOP in hydroxyl radical production as measured by adenine dosimetry and the ability to heavily modify proteins without labeling oxidatively altered conformations. 18 Here, we both investigated important figures of merit of the FOX system and tested its ability to generate reliable and useful structural results by mapping the adalimumab epitope on TNFα. Laser-based FPOP is generally considered to have a primary labeling lifetime of ∼1 μs, 9 although secondary radicals almost certainly persist much longer 25 and may result in a slower oxidation of sulfur-containing residues.…”

Section: ■ Conclusionmentioning

confidence: 99%

“…It has also been shown to be fast enough to label proteins without oxidationinduced conformational changes based on previously published measurement of dose-dependent labeling. 18 The flow injection sample introduction system is a significant departure from the syringe pump delivery used by the traditional FPOP. The flow injection method allows for more reproducible sample delivery and a faster sample-tosample transition and ensures thorough capillary cleaning between samples to minimize carry-over.…”

Section: ■ Conclusionmentioning

confidence: 99%

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

Sharp

Chea²,

Misra

et al. 2021

J. Am. Soc. Mass Spectrom.

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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.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: ■ Conclusionmentioning

confidence: 99%

Section: ■ Conclusionmentioning

confidence: 99%

See 3 more Smart Citations

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

Sharp

Chea²,

Misra

et al. 2021

J. Am. Soc. Mass Spectrom.

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Advances in mass spectrometry-based epitope mapping of protein therapeutics

Liu

Huang

Zhao

et al. 2022

Journal of Pharmaceutical and Biomedical Analysis

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Structural Analysis of Phosphorylation Proteoforms in a Dynamic Heterogeneous System Using Flash Oxidation Coupled In-Line with Ion Exchange Chromatography

et al. 2022

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Protein posttranslational modifications (PTMs) are key modulators of protein structure and function that often change in a dynamic fashion in response to cellular stimuli. Dynamic PTMs are very challenging to structurally characterize using modern techniques, including covalent labeling methods, due to the presence of multiple proteoforms and conformers together in solution. We have coupled an ion exchange high-performance liquid chromatography separation with a flash oxidation system [ion exchange chromatography liquid chromatography-flash oxidation (IEX LC-FOX)] to successfully elucidate structural changes among three phosphoproteoforms of ovalbumin (OVA) during dephosphorylation with alkaline phosphatase. Real-time dosimetry indicates no difference in the effective radical dose between peaks or across the peak, demonstrating both the lack of scavenging of the NaCl gradient and the lack of a concentration effect on radical dose between peaks of different intensities. The use of IEX LC-FOX allows us to structurally probe into each phosphoproteoform as it elutes from the column, capturing structural data before the dynamics of the system to reintroduce heterogeneity. We found significant differences in the residue-level oxidation between the hydroxyl radical footprint of nonphosphorylated, monophosphorylated, and diphosphorylated OVA. Not only were our data consistent with the previously reported stabilization of OVA structure by phosphorylation, but local structural changes were also consistent with the measured order of dephosphorylation of Ser344 being removed first. These results demonstrate the utility of IEX LC-FOX for measuring the structural effects of PTMs, even in dynamic systems.

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Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins

Cited by 8 publications

References 33 publications

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

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

Advances in mass spectrometry-based epitope mapping of protein therapeutics

Structural Analysis of Phosphorylation Proteoforms in a Dynamic Heterogeneous System Using Flash Oxidation Coupled In-Line with Ion Exchange Chromatography

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