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

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, unexpectedly, as a protein complex with a molecular weight of 211 kDa with no homology to known proteins in the database. 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 experiment led to a plausible model of this unknown multicopper oxidase which has been difficult to study by conventional structural biology methods. However, because each subunit of Mnx binds copper ions as cofactor at varying ratios, there were remaining ambiguities in assigning some of the observed peaks to metal-binding species because of the sample heterogeneity and limited mass resolution. In this study, we performed SID in a modified Fourier transform – ion cyclotron resonance (FT-ICR) mass spectrometer for obtaining the ultimate resolution on the released subcomplexes of Mnx. The high mass accuracy and resolution unveiled unexpectedmore » artificial modifications in the protein that have been previously thought to be iron bound species based on lower resolution data. Additionally, most released subcomplexes were isotopically resolved for defining metal binding stoichiometry at each structural level. This method holds great potential for in-depth characterization of metalloproteins and protein-ligand complexes.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1439695
Report Number(s):
PNNL-SA-124854
Journal ID: ISSN 1044-0305; 47955; 48903; KP1704020
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Journal of the American Society for Mass Spectrometry
Additional Journal Information:
Journal Volume: 29; Journal Issue: 4; Journal ID: ISSN 1044-0305
Publisher:
American Society for Mass Spectrometry
Country of Publication:
United States
Language:
English
Subject:
Native Mass Spectrometry; high resolution mass spectrometry; protein complex; surface induced dissociation; metalloprotein; protein-ligand interaction; Environmental Molecular Sciences Laboratory

Citation Formats

Zhou, Mowei, Yan, Jing, Romano, Christine A., Tebo, Bradley M., Wysocki, Vicki H., and Paša-Tolić, Ljiljana. Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex. United States: N. p., 2018. Web. doi:10.1007/s13361-017-1882-x.
Zhou, Mowei, Yan, Jing, Romano, Christine A., Tebo, Bradley M., Wysocki, Vicki H., & Paša-Tolić, Ljiljana. Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex. United States. doi:10.1007/s13361-017-1882-x.
Zhou, Mowei, Yan, Jing, Romano, Christine A., Tebo, Bradley M., Wysocki, Vicki H., and Paša-Tolić, Ljiljana. Wed . "Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex". United States. doi:10.1007/s13361-017-1882-x.
@article{osti_1439695,
title = {Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex},
author = {Zhou, Mowei and Yan, Jing and Romano, Christine A. and Tebo, Bradley M. and Wysocki, Vicki H. and Paša-Tolić, Ljiljana},
abstractNote = {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, unexpectedly, as a protein complex with a molecular weight of 211 kDa with no homology to known proteins in the database. 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 experiment led to a plausible model of this unknown multicopper oxidase which has been difficult to study by conventional structural biology methods. However, because each subunit of Mnx binds copper ions as cofactor at varying ratios, there were remaining ambiguities in assigning some of the observed peaks to metal-binding species because of the sample heterogeneity and limited mass resolution. In this study, we performed SID in a modified Fourier transform – ion cyclotron resonance (FT-ICR) mass spectrometer for obtaining the ultimate resolution on the released subcomplexes of Mnx. The high mass accuracy and resolution unveiled unexpected artificial modifications in the protein that have been previously thought to be iron bound species based on lower resolution data. Additionally, most released subcomplexes were isotopically resolved for defining metal binding stoichiometry at each structural level. This method holds great potential for in-depth characterization of metalloproteins and protein-ligand complexes.},
doi = {10.1007/s13361-017-1882-x},
journal = {Journal of the American Society for Mass Spectrometry},
issn = {1044-0305},
number = 4,
volume = 29,
place = {United States},
year = {2018},
month = {1}
}

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