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Expanding the mass range for UVPD-based native top-down mass spectrometry.
Journal article

Expanding the mass range for UVPD-based native top-down mass spectrometry.

  • Greisch JF Biomolecular Mass Spectrometry and Proteomics , Bijvoet Center for Biomolecular Research , Utrecht Institute of Pharmaceutical Sciences , Utrecht University , Padualaan 8 , 3584 Utrecht , The Netherlands . Email: a.j.r.heck@uu.nl.
  • Tamara S Biomolecular Mass Spectrometry and Proteomics , Bijvoet Center for Biomolecular Research , Utrecht Institute of Pharmaceutical Sciences , Utrecht University , Padualaan 8 , 3584 Utrecht , The Netherlands . Email: a.j.r.heck@uu.nl.
  • Scheltema RA Biomolecular Mass Spectrometry and Proteomics , Bijvoet Center for Biomolecular Research , Utrecht Institute of Pharmaceutical Sciences , Utrecht University , Padualaan 8 , 3584 Utrecht , The Netherlands . Email: a.j.r.heck@uu.nl.
  • Maxwell HWR Department of Microbiology and Immunology , University of Otago , PO Box 56 , 9054 Dunedin , New Zealand.
  • Fagerlund RD Department of Microbiology and Immunology , University of Otago , PO Box 56 , 9054 Dunedin , New Zealand.
  • Fineran PC Department of Microbiology and Immunology , University of Otago , PO Box 56 , 9054 Dunedin , New Zealand.
  • Tetter S Laboratory of Organic Chemistry , Department of Chemistry and Applied Biosciences , ETH Zürich , Vladimir-Prelog-Weg 1-5/10 , 8093 Zürich , Switzerland.
  • Hilvert D Laboratory of Organic Chemistry , Department of Chemistry and Applied Biosciences , ETH Zürich , Vladimir-Prelog-Weg 1-5/10 , 8093 Zürich , Switzerland.
  • Heck AJR Biomolecular Mass Spectrometry and Proteomics , Bijvoet Center for Biomolecular Research , Utrecht Institute of Pharmaceutical Sciences , Utrecht University , Padualaan 8 , 3584 Utrecht , The Netherlands . Email: a.j.r.heck@uu.nl.
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  • 2019-10-08
Published in:
  • Chemical science. - 2019
General Chemistry
English Native top-down mass spectrometry is emerging as a methodology that can be used to structurally investigate protein assemblies. To extend the possibilities of native top-down mass spectrometry to larger and more heterogeneous biomolecular assemblies, advances in both the mass analyzer and applied fragmentation techniques are still essential. Here, we explore ultraviolet photodissociation (UVPD) of protein assemblies on an Orbitrap with extended mass range, expanding its usage to large and heterogeneous macromolecular complexes, reaching masses above 1 million Da. We demonstrate that UVPD can lead not only to the ejection of intact subunits directly from such large intact complexes, but also to backbone fragmentation of these subunits, providing enough sequence information for subunit identification. The Orbitrap mass analyzer enables simultaneous monitoring of the precursor, the subunits, and the subunit fragments formed upon UVPD activation. While only partial sequence coverage of the subunits is observed, the UVPD data yields information about the localization of chromophores covalently attached to the subunits of the light harvesting complex B-phycoerythrin, extensive backbone fragmentation in a subunit of a CRISPR-Cas Csy (type I-F Cascade) complex, and sequence modifications in a virus-like proteinaceous nano-container. Through these multiple applications we demonstrate for the first time that UVPD based native top-down mass spectrometry is feasible for large and heterogeneous particles, including ribonucleoprotein complexes and MDa virus-like particles.
Language
  • English
Open access status
gold
Identifiers
Persistent URL
https://folia.unifr.ch/global/documents/200483
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