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Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex

  • Mowei Zhou
  • Jing Yan
  • Christine A. Romano
  • Bradley M. Tebo
  • Vicki H. Wysocki
  • Ljiljana Paša-Tolić
Article

Abstract

Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese oxidase, Mnx, from Bacillus sp. PL-12 has been successfully overexpressed in active form as a protein complex with a molecular mass of 211 kDa. We have recently used surface induced dissociation (SID) and ion mobility-mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data from the native mass spectrometry experiments led to a plausible structural model of this multicopper oxidase, which has been difficult to study by conventional structural biology methods. It was also revealed that each Mnx subunit binds a variable number of copper ions. Becasue of the heterogeneity of the protein and limited mass resolution, ambiguities in assigning some of the observed peaks remained as a barrier to fully understanding the role of metals and potential unknown ligands in Mnx. In this study, we performed SID in a modified Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer. The high mass accuracy and resolution offered by FTICR unveiled unexpected artificial modifications on the protein that had been previously thought to be iron bound species based on lower resolution spectra. Additionally, isotopically resolved spectra of the released subcomplexes revealed the metal binding stoichiometry at different structural levels. This method holds great potential for in-depth characterization of metalloproteins and protein–ligand complexes.

Graphical Abstract

Keywords

Native mass spectrometry High resolution mass spectrometry Protein complex Surface induced dissociation Metalloprotein Protein–ligand interaction 

Notes

Acknowledgments

The authors thank Yang Song and Arpad Somogyi at The Ohio State University for helping with the SID experiments; Jeremy Wolff and Michael Easterling at Bruker Corporation, Randy Pedder at Ardara Technologies for helping with the instrument modification. This work was funded by the National Science Foundation (NSF DBI 1455654; SID development and installation) and National Institute of Health (NIH 1S10OD018507; FTICR purchase) to V.H.W., NSF CHE-1410688 to B.M.T, and an NSF Postdoctoral Research Fellowship in Biology Award ID: DBI-1202859 to C.A.R. A portion of the research was supported by the Environmental and Molecular Sciences Laboratory (EMSL), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory.

Supplementary material

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Copyright information

© American Society for Mass Spectrometry (outside the USA) 2018

Authors and Affiliations

  • Mowei Zhou
    • 1
  • Jing Yan
    • 2
  • Christine A. Romano
    • 3
  • Bradley M. Tebo
    • 3
  • Vicki H. Wysocki
    • 2
  • Ljiljana Paša-Tolić
    • 1
  1. 1.Environmental Molecular Sciences Laboratory, Pacific Northwest National LaboratoryRichlandUSA
  2. 2.Department of Chemistry and BiochemistryOhio State UniversityColumbusUSA
  3. 3.Division of Environmental and Biomolecular SystemsOregon Health & Science UniversityPortlandUSA

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