ArticleJournal of the American Society for Mass Spectrometry2016
Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds.
Article in Journal of the American Society for Mass Spectrometry, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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30 citing papers in PubMed, 47 citations in OpenAlex.
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- Fenton-Chemistry-Based Oxidative Modification of Proteins Reflects Their Conformation.International journal of molecular sciences · 2021Article
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- Flash Oxidation (FOX) System: A Novel Laser-Free Fast Photochemical Oxidation Protein Footprinting Platform.Journal of the American Society for Mass Spectrometry · 2021Article
- Self-Organized Amphiphiles Are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments.Journal of the American Society for Mass Spectrometry · 2021Article
- Structural Proteomics Methods to Interrogate the Conformations and Dynamics of Intrinsically Disordered Proteins.Frontiers in chemistry · 2021Review
- Chemical Penetration Enhancers Increase Hydrogen Peroxide Uptake inJournal of proteome research · 2020Article
- Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes.Journal of visualized experiments : JoVE · 2020Article
- Parallel Chemoselective Profiling for Mapping Protein Structure.Cell chemical biology · 2020Article
- Mass spectrometry-based methods in characterization of the higher order structure of protein therapeutics.Journal of pharmaceutical and biomedical analysis · 2020Review
- Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications.Chemical reviews · 2020Review
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Hydroxyl radical (⋅OH) labeling with mass spectrometry detection reports on protein conformations and interactions. Fast photochemical oxidation of proteins (FPOP) involves ⋅OH production via H2O2 photolysis by UV laser pulses inside a flow tube. The experiments are conducted in the presence of a scavenger (usually glutamine) that shortens the ⋅OH lifetime. The literature claims that FPOP takes place within 1 μs. This ultrafast time scale implies that FPOP should be immune to labeling-induced artifacts that may be encountered with other techniques. Surprisingly, the FPOP time scale has never been validated in direct kinetic measurements. Here we employ flash photolysis for probing oxidation processes under typical FPOP conditions. Bleaching of the reporter dye cyanine-5 (Cy5) served as readout of the time-dependent radical milieu. Surprisingly, Cy5 oxidation extends over tens of milliseconds. This time range is four orders of magnitude longer than expected from the FPOP literature. We demonstrate that the glutamine scavenger generates metastable secondary radicals in the FPOP solution, and that these radicals lengthen the time frame of Cy5 oxidation. Cy5 and similar dyes are widely used for monitoring the radical dose experienced by proteins in solution. The measured Cy5 kinetics thus strongly suggest that protein oxidation in FPOP extends over a much longer time window than previously thought (i.e., many milliseconds instead of one microsecond). The optical approach developed here should be suitable for assessing the performance of future FPOP-like techniques with improved temporal labeling characteristics. Graphical Abstract ᅟ.
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