Journal Article

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

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

2018

Overview

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 ᅟ

• Publisher: Springer US,Springer Nature B.V,American Society for Mass Spectrometry
• Subject: Analytical Chemistry / Bacteria / Bioinformatics / Biotechnology / Chemistry / Chemistry and Materials Science / Coordination compounds / Cyclotron resonance / Environmental Molecular Sciences Laboratory / Fourier transforms / Heterogeneity / high resolution mass spectrometry / Ionic mobility / Ions / Ligands / Manganese / Mass spectrometry / metalloprotein / Native Mass Spectrometry / Organic Chemistry / Oxidase / Oxidation / protein complex / protein-ligand interaction / Proteins / Proteomics / Scientific imaging / Spectroscopy / surface induced dissociation