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Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry
In the method of X-ray footprinting mass spectrometry (XFMS), proteins at micromolar concentration in solution are irradiated with a broadband X-ray source, and the resulting hydroxyl radical modifications are characterized using liquid chromatography mass spectrometry to determine sites of solvent...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
International Union of Crystallography
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8415330/ https://www.ncbi.nlm.nih.gov/pubmed/34475282 http://dx.doi.org/10.1107/S1600577521004744 |
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author | Kristensen, Line G. Holton, James M. Rad, Behzad Chen, Yan Petzold, Christopher J. Gupta, Sayan Ralston, Corie Y. |
author_facet | Kristensen, Line G. Holton, James M. Rad, Behzad Chen, Yan Petzold, Christopher J. Gupta, Sayan Ralston, Corie Y. |
author_sort | Kristensen, Line G. |
collection | PubMed |
description | In the method of X-ray footprinting mass spectrometry (XFMS), proteins at micromolar concentration in solution are irradiated with a broadband X-ray source, and the resulting hydroxyl radical modifications are characterized using liquid chromatography mass spectrometry to determine sites of solvent accessibility. These data are used to infer structural changes in proteins upon interaction with other proteins, folding, or ligand binding. XFMS is typically performed under aerobic conditions; dissolved molecular oxygen in solution is necessary in many, if not all, the hydroxyl radical modifications that are generally reported. In this study we investigated the result of X-ray induced modifications to three different proteins under aerobic versus low oxygen conditions, and correlated the extent of damage with dose calculations. We observed a concentration-dependent protecting effect at higher protein concentration for a given X-ray dose. For the typical doses used in XFMS experiments there was minimal X-ray induced aggregation and fragmentation, but for higher doses we observed formation of covalent higher molecular weight oligomers, as well as fragmentation, which was affected by the amount of dissolved oxygen in solution. The higher molecular weight products in the form of dimers, trimers, and tetramers were present in all sample preparations, and, upon X-ray irradiation, these oligomers became non-reducible as seen in SDS-PAGE. The results provide an important contribution to the large body of X-ray radiation damage literature in structural biology research, and will specifically help inform the future planning of XFMS, and well as X-ray crystallography and small-angle X-ray scattering experiments. |
format | Online Article Text |
id | pubmed-8415330 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | International Union of Crystallography |
record_format | MEDLINE/PubMed |
spelling | pubmed-84153302021-09-16 Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry Kristensen, Line G. Holton, James M. Rad, Behzad Chen, Yan Petzold, Christopher J. Gupta, Sayan Ralston, Corie Y. J Synchrotron Radiat Radiation Damage In the method of X-ray footprinting mass spectrometry (XFMS), proteins at micromolar concentration in solution are irradiated with a broadband X-ray source, and the resulting hydroxyl radical modifications are characterized using liquid chromatography mass spectrometry to determine sites of solvent accessibility. These data are used to infer structural changes in proteins upon interaction with other proteins, folding, or ligand binding. XFMS is typically performed under aerobic conditions; dissolved molecular oxygen in solution is necessary in many, if not all, the hydroxyl radical modifications that are generally reported. In this study we investigated the result of X-ray induced modifications to three different proteins under aerobic versus low oxygen conditions, and correlated the extent of damage with dose calculations. We observed a concentration-dependent protecting effect at higher protein concentration for a given X-ray dose. For the typical doses used in XFMS experiments there was minimal X-ray induced aggregation and fragmentation, but for higher doses we observed formation of covalent higher molecular weight oligomers, as well as fragmentation, which was affected by the amount of dissolved oxygen in solution. The higher molecular weight products in the form of dimers, trimers, and tetramers were present in all sample preparations, and, upon X-ray irradiation, these oligomers became non-reducible as seen in SDS-PAGE. The results provide an important contribution to the large body of X-ray radiation damage literature in structural biology research, and will specifically help inform the future planning of XFMS, and well as X-ray crystallography and small-angle X-ray scattering experiments. International Union of Crystallography 2021-07-20 /pmc/articles/PMC8415330/ /pubmed/34475282 http://dx.doi.org/10.1107/S1600577521004744 Text en © Line G. Kristensen et al. 2021 https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. |
spellingShingle | Radiation Damage Kristensen, Line G. Holton, James M. Rad, Behzad Chen, Yan Petzold, Christopher J. Gupta, Sayan Ralston, Corie Y. Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry |
title | Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry |
title_full | Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry |
title_fullStr | Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry |
title_full_unstemmed | Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry |
title_short | Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry |
title_sort | hydroxyl radical mediated damage of proteins in low oxygen solution investigated using x-ray footprinting mass spectrometry |
topic | Radiation Damage |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8415330/ https://www.ncbi.nlm.nih.gov/pubmed/34475282 http://dx.doi.org/10.1107/S1600577521004744 |
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