Impact of limited oxidation on protein ion mobility and structure of importance to footprinting by radical probe mass spectrometry

Downard, Kevin M.; Maleknia, Simin D.; Akashi, Satoko

doi:10.1002/rcm.5320

Cited by 25 publications

(31 citation statements)

References 33 publications

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“…Higher levels of M+n16 (n>1) Da ubiquitin relative to unmodified species have been shown with other oxidizing methods. 35–38 …”

Section: Resultsmentioning

confidence: 99%

“…38 The observed structures for monooxidized and unmodified ions were compact in conformation for +5 ions. 38 The distributions for ions of charge state +5 were identical for monooxidized and unmodified ions based on a single compact conformer, whereas ions of charge state +6 favored the more compact structure of two observed conformers for monooxidized species. Those IMS-MS experiments were performed on a travelling-wave IMS-MS instrument and reported no significant change to ubiquitin structures due to oxidation.…”

Section: Introductionmentioning

confidence: 91%

“…Those IMS-MS experiments were performed on a travelling-wave IMS-MS instrument and reported no significant change to ubiquitin structures due to oxidation. 38 The work presented here seeks to provide further insight and clarification to the effects of oxidation on ubiquitin conformers.…”

Section: Introductionmentioning

confidence: 99%

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Effects of Fe(II)/H₂O₂ Oxidation on Ubiquitin Conformers Measured by Ion Mobility-Mass Spectrometry

Shi

Clemmer

et al. 2012

J. Phys. Chem. B

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Oxidative modifications can have significant effects on protein structure in solution. Here, the structures and stabilities of oxidized ubiquitin ions electrosprayed from an aqueous solution (pH 2) are studied by ion mobility spectrometry-mass spectrometry (IMS-MS). IMS-MS has proven to be a valuable technique to assess gas-phase and in many cases, solution structures. Herein, in vitro oxidation is performed by Fenton chemistry with Fe(II)/hydrogen peroxide. Most molecules in solution remain unmodified whereas ~20% of the population belongs to an M+16 Da oxidized species. Ions of low charge states (+7 and +8) show substantial variance in collision cross section distributions between unmodified and oxidized species. Novel and previously reported Gaussian conformers are used to model cross section distributions for +7 and +8 oxidized ubiquitin ions, respectively, in order to correlate variances in observed gas-phase distributions to changes in populations of solution states. Based on Gaussian modeling, oxidized ions of charge state +7 have an A-state conformation which is more populated for oxidized relative to unmodified ions. Oxidized ubiquitin ions of charge state +8 have a distribution of conformers arising from native-state ubiquitin and higher intensities of A- and U-state conformers relative to unmodified ions. This work provides evidence that incorporation of a single oxygen atom to ubiquitin leads to destabilization of the native state in an acidic solution (pH ~2) and to unfolding of gas-phase compact structures.

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“…Higher levels of M+n16 (n>1) Da ubiquitin relative to unmodified species have been shown with other oxidizing methods. 35–38 …”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 91%

See 1 more Smart Citation

Effects of Fe(II)/H₂O₂ Oxidation on Ubiquitin Conformers Measured by Ion Mobility-Mass Spectrometry

Shi

Clemmer

et al. 2012

J. Phys. Chem. B

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“…However, any change in protein concentration would impact oxidation kinetics due to altered match with flux of hydroxyl radicals over the experimental timeframe 23 and interfere with structural interpretation of circular dichroism (CD) results. 24 Two of these model proteins were already shown to be structurally resilient to oxidative damage by CD measurements and intact protein mass spectrometry, 25 so the latter metric alone was used for gauging structural integrity.…”

Section: Resultsmentioning

confidence: 99%

Experimental Approach to Controllably Vary Protein Oxidation While Minimizing Electrode Adsorption for Boron-Doped Diamond Electrochemical Surface Mapping Applications

McClintock

Hettich

2012

Anal. Chem.

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Oxidative protein surface mapping has become a powerful approach for measuring the solvent accessibility of folded protein structures. A variety of techniques exist for generating the key reagent – hydroxyl radicals – for these measurements; however, these approaches range significantly in their complexity and expense of operation. This research expands upon earlier work to enhance the controllability of boron-doped diamond (BDD) electrochemistry as an easily accessible tool for producing hydroxyl radicals in order to oxidize a range of intact proteins. Efforts to modulate oxidation level while minimizing the adsorption of protein to the electrode involved the use of relatively high flow rates to reduce protein residence time inside the electrochemical flow chamber. Additionally, a different cell activation approach using variable voltage to supply a controlled current allowed us to precisely tune the extent of oxidation in a protein-dependent manner. In order to gain perspective on the level of protein adsorption onto the electrode surface, studies were conducted to monitor protein concentration during electrolysis and gauge changes in the electrode surface between cell activation events. This report demonstrates the successful use of BDD electrochemistry for greater precision in generating a target number of oxidation events upon intact proteins.

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“…6 Hydroxyl radical footprinting of proteins, 7 also known as radical probe mass spectrometry 8 or oxidative protein surface mapping, 9 presents a unique challenge to both experimental and computational strategies, because multiple oxidation events can occur on virtually any amino acid. 10 The substantial increase in sample complexity induced by oxidation can potentially generate a wide dynamic range of peptide abundances, as the mixture is often dominated by the unmodified versions of oxidized peptide variants.…”

Section: Introductionmentioning

confidence: 99%

Comparative Informatics Analysis to Evaluate Site-Specific Protein Oxidation in Multidimensional LC–MS/MS Data

et al. 2013

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Redox proteomics has yielded molecular insight into diseases of protein dysfunction attributable to oxidative stress, underscoring the need for robust detection of protein oxidation products. Additionally, oxidative protein surface mapping techniques utilize hydroxyl radicals to gain structural insight about solvent exposure. Interpretation of tandem mass spectral data is a critical challenge for such investigations, because reactive oxygen species target a wide breadth of amino acids. Additionally, oxidized peptides may be generated in a wide range of abundances since the reactivity of hydroxyl radicals with different amino acids spans three orders of magnitude. Taken together, these attributes of oxidative footprinting pose both experimental and computational challenges to detecting oxidized peptides that are naturally less abundant than their unoxidized counterparts. In this study, three model proteins were oxidized electrochemically and analyzed at both the intact protein and peptide levels. A multidimensional chromatographic strategy was utilized to expand the dynamic range of oxidized peptide measurements. Peptide mass spectral data were searched by the “hybrid” software packages Inspect and Byonic, which incorporate de novo elements of spectral interpretation into a database search. This dynamic search capacity accommodates the challenge of searching for more than forty oxidative mass shifts that can occur in a staggering variety of possible combinatorial occurrences. A prevailing set of oxidized residues was identified with this comparative approach, and evaluation of these sites was informed by solvent accessible surface area gleaned through molecular dynamics simulations. Along with increased levels of oxidation around highly reactive “hotspot” sites as expected, the enhanced sensitivity of these measurements uncovered a surprising level of oxidation on less reactive residues.

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Impact of limited oxidation on protein ion mobility and structure of importance to footprinting by radical probe mass spectrometry

Cited by 25 publications

References 33 publications

Effects of Fe(II)/H₂O₂ Oxidation on Ubiquitin Conformers Measured by Ion Mobility-Mass Spectrometry

Effects of Fe(II)/H₂O₂ Oxidation on Ubiquitin Conformers Measured by Ion Mobility-Mass Spectrometry

Experimental Approach to Controllably Vary Protein Oxidation While Minimizing Electrode Adsorption for Boron-Doped Diamond Electrochemical Surface Mapping Applications

Comparative Informatics Analysis to Evaluate Site-Specific Protein Oxidation in Multidimensional LC–MS/MS Data

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Impact of limited oxidation on protein ion mobility and structure of importance to footprinting by radical probe mass spectrometry

Cited by 25 publications

References 33 publications

Effects of Fe(II)/H2O2 Oxidation on Ubiquitin Conformers Measured by Ion Mobility-Mass Spectrometry

Effects of Fe(II)/H2O2 Oxidation on Ubiquitin Conformers Measured by Ion Mobility-Mass Spectrometry

Experimental Approach to Controllably Vary Protein Oxidation While Minimizing Electrode Adsorption for Boron-Doped Diamond Electrochemical Surface Mapping Applications

Comparative Informatics Analysis to Evaluate Site-Specific Protein Oxidation in Multidimensional LC–MS/MS Data

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Effects of Fe(II)/H₂O₂ Oxidation on Ubiquitin Conformers Measured by Ion Mobility-Mass Spectrometry

Effects of Fe(II)/H₂O₂ Oxidation on Ubiquitin Conformers Measured by Ion Mobility-Mass Spectrometry