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Quantifying the heterogeneity of macromolecular machines by mass photometry
Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the hete...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156492/ https://www.ncbi.nlm.nih.gov/pubmed/32286308 http://dx.doi.org/10.1038/s41467-020-15642-w |
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author | Sonn-Segev, Adar Belacic, Katarina Bodrug, Tatyana Young, Gavin VanderLinden, Ryan T. Schulman, Brenda A. Schimpf, Johannes Friedrich, Thorsten Dip, Phat Vinh Schwartz, Thomas U. Bauer, Benedikt Peters, Jan-Michael Struwe, Weston B. Benesch, Justin L. P. Brown, Nicholas G. Haselbach, David Kukura, Philipp |
author_facet | Sonn-Segev, Adar Belacic, Katarina Bodrug, Tatyana Young, Gavin VanderLinden, Ryan T. Schulman, Brenda A. Schimpf, Johannes Friedrich, Thorsten Dip, Phat Vinh Schwartz, Thomas U. Bauer, Benedikt Peters, Jan-Michael Struwe, Weston B. Benesch, Justin L. P. Brown, Nicholas G. Haselbach, David Kukura, Philipp |
author_sort | Sonn-Segev, Adar |
collection | PubMed |
description | Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization. |
format | Online Article Text |
id | pubmed-7156492 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-71564922020-04-22 Quantifying the heterogeneity of macromolecular machines by mass photometry Sonn-Segev, Adar Belacic, Katarina Bodrug, Tatyana Young, Gavin VanderLinden, Ryan T. Schulman, Brenda A. Schimpf, Johannes Friedrich, Thorsten Dip, Phat Vinh Schwartz, Thomas U. Bauer, Benedikt Peters, Jan-Michael Struwe, Weston B. Benesch, Justin L. P. Brown, Nicholas G. Haselbach, David Kukura, Philipp Nat Commun Article Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization. Nature Publishing Group UK 2020-04-14 /pmc/articles/PMC7156492/ /pubmed/32286308 http://dx.doi.org/10.1038/s41467-020-15642-w Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Sonn-Segev, Adar Belacic, Katarina Bodrug, Tatyana Young, Gavin VanderLinden, Ryan T. Schulman, Brenda A. Schimpf, Johannes Friedrich, Thorsten Dip, Phat Vinh Schwartz, Thomas U. Bauer, Benedikt Peters, Jan-Michael Struwe, Weston B. Benesch, Justin L. P. Brown, Nicholas G. Haselbach, David Kukura, Philipp Quantifying the heterogeneity of macromolecular machines by mass photometry |
title | Quantifying the heterogeneity of macromolecular machines by mass photometry |
title_full | Quantifying the heterogeneity of macromolecular machines by mass photometry |
title_fullStr | Quantifying the heterogeneity of macromolecular machines by mass photometry |
title_full_unstemmed | Quantifying the heterogeneity of macromolecular machines by mass photometry |
title_short | Quantifying the heterogeneity of macromolecular machines by mass photometry |
title_sort | quantifying the heterogeneity of macromolecular machines by mass photometry |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156492/ https://www.ncbi.nlm.nih.gov/pubmed/32286308 http://dx.doi.org/10.1038/s41467-020-15642-w |
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