Quantifying the heterogeneity of macromolecular machines by mass photometry

Adar Sonn-Segev, Katarina Belacic, Tatyana Bodrug, Gavin Young, Ryan T. VanderLinden, Brenda A. Schulman, Johannes Schimpf, Thorsten Friedrich, Phat Vinh Dip, Thomas U. Schwartz, Benedikt Bauer, Jan-Michael Peters, Weston B. Struwe, Justin L. P. Benesch, Nicholas G. Brown (), David Haselbach () and Philipp Kukura ()
Additional contact information
Adar Sonn-Segev: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford
Katarina Belacic: Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC)
Tatyana Bodrug: Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill
Gavin Young: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford
Ryan T. VanderLinden: Department of Structural Biology, St. Jude Children’s Research Hospital
Brenda A. Schulman: Department of Structural Biology, St. Jude Children’s Research Hospital
Johannes Schimpf: Albert-Ludwigs-Universität, Institut für Biochemie, Albertstr. 21, Chemie-Hochhaus
Thorsten Friedrich: Albert-Ludwigs-Universität, Institut für Biochemie, Albertstr. 21, Chemie-Hochhaus
Phat Vinh Dip: Department of Biology, Massachusetts Institute of Technology
Thomas U. Schwartz: Department of Biology, Massachusetts Institute of Technology
Benedikt Bauer: Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC)
Jan-Michael Peters: Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC)
Weston B. Struwe: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford
Justin L. P. Benesch: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford
Nicholas G. Brown: Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine
David Haselbach: Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC)
Philipp Kukura: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford

Nature Communications, 2020, vol. 11, issue 1, 1-10

Abstract: Abstract 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.

Date: 2020
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DOI: 10.1038/s41467-020-15642-w

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