Fenton-Chemistry-Based Oxidative Modification of Proteins Reflects Their Conformation

Abstract

In order to understand protein structure to a sufficient extent for, e.g., drug discovery, no single technique can provide satisfactory information on both the lowest-energy conformation and on dynamic changes over time (the ‘four-dimensional’ protein structure). Instead, a combination of complementary techniques is required. Mass spectrometry methods have shown promise in addressing protein dynamics, but often rely on the use of high-end commercial or custom instruments. Here, we apply well-established chemistry to conformation-sensitive oxidative protein labelling on a timescale of a few seconds, followed by analysis through a routine protein analysis workflow. For a set of model proteins, we show that site selectivity of labelling can indeed be rationalised in terms of known structural information, and that conformational changes induced by ligand binding are reflected in the modification pattern. In addition to conventional bottom-up analysis, further insights are obtained from intact mass measurement and native mass spectrometry. We believe that this method will provide a valuable and robust addition to the ‘toolbox’ of mass spectrometry researchers studying higher-order protein structure.

Unveiling Molecular Transformations of Soil Organic Matter after Remediation by Chemical Oxidation Based on ESI-FT-ICR-MS analysis

“…However, such organic consumption would reduce the degradation efficiency of diesels by competing with •OH. In addition, the intensity magnification of tyrosine-like protein after FT treatment may be ascribed to the hydrolysis of macromolecular proteins . On the other hand, after BP treatment, the content of FAL and protein-like substances (including tyrosine-like and tryptophan-like proteins) in BPS decreased, which was attributed to these substances having high reactivity in oxidation .…”

Systematic Fe(II)-EDTA Method of Dose-Dependent Hydroxyl Radical Generation for Protein Oxidative Footprinting

“…In contrast, oxidizing 96 samples one by one using Fe­(II)-EDTA/H 2 O 2 /ascorbate (e.g., ref ) would take hours. This length of time exceeds the stability of the Fe­(II)-EDTA reagent (Figure S1I) and that of ascorbate and likely the endurance of all but the hardiest scientist. (iii) We have demonstrated that chemical generation of • OH is compatible with a range of solutes and conditions commonly used to analyze proteins (Figure S1).…”

“…That Fenton-generated • OH can be used for protein oxidative footprinting has been demonstrated. − These studies utilized the Tullius nucleic acid analysis approach of adding Fe­(II)-EDTA, H 2 O 2 , and ascorbate to generate • OH in samples. While nucleic acid oxidative footprinting studies are typically conducted using a single radical dose, the importance of dose-dependent oxidation to protein analysis is well established. , We found that the Fe­(II)-EDTA/H 2 O 2 /ascorbate cocktail does not support the reproducible dose-dependent generation of • OH required for reliable protein analysis.…”

Detection and Verification of a Key Intermediate in an Enantioselective Peptide Catalyzed Acylation Reaction

“…Fits of the reaction profile using zeroth, first and second order kinetics (Figure S17; Figure S16; Figure S18). Ref [ 11 , 17 , 22 , 26 , 27 , 28 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ] are cited in the Supplementary Materials.…”