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...
| Main Authors: | , , , , , , , , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
Springer Nature
2020
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| Subjects: |
| _version_ | 1826295572797063168 |
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| author | Sonn-Segev, A Belacic, K Bodrug, T Young, G VanderLinden, RT Schulman, BA Schimpf, J Friedrich, T Dip, PV Schwartz, TU Bauer, B Peters, J-M Benesch, JLP Struwe, WB Brown, NG Haselbach, D Kukura, P |
| author_facet | Sonn-Segev, A Belacic, K Bodrug, T Young, G VanderLinden, RT Schulman, BA Schimpf, J Friedrich, T Dip, PV Schwartz, TU Bauer, B Peters, J-M Benesch, JLP Struwe, WB Brown, NG Haselbach, D Kukura, P |
| author_sort | Sonn-Segev, A |
| collection | OXFORD |
| 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. |
| first_indexed | 2024-03-07T04:03:07Z |
| format | Journal article |
| id | oxford-uuid:c543fc6c-3a3c-4a33-ae12-5f69639e4e21 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T04:03:07Z |
| publishDate | 2020 |
| publisher | Springer Nature |
| record_format | dspace |
| spelling | oxford-uuid:c543fc6c-3a3c-4a33-ae12-5f69639e4e212022-03-27T06:29:36ZQuantifying the heterogeneity of macromolecular machines by mass photometryJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:c543fc6c-3a3c-4a33-ae12-5f69639e4e21Cryoelectron microscopyStructural biologySingle-molecule biophysicsHigh-throughput screeningEnglishSymplectic ElementsSpringer Nature2020Sonn-Segev, ABelacic, KBodrug, TYoung, GVanderLinden, RTSchulman, BASchimpf, JFriedrich, TDip, PVSchwartz, TUBauer, BPeters, J-MBenesch, JLPStruwe, WBBrown, NGHaselbach, DKukura, PSample 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. |
| spellingShingle | Cryoelectron microscopy Structural biology Single-molecule biophysics High-throughput screening Sonn-Segev, A Belacic, K Bodrug, T Young, G VanderLinden, RT Schulman, BA Schimpf, J Friedrich, T Dip, PV Schwartz, TU Bauer, B Peters, J-M Benesch, JLP Struwe, WB Brown, NG Haselbach, D Kukura, P 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 | Cryoelectron microscopy Structural biology Single-molecule biophysics High-throughput screening |
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