Identification of metal-binding sites in proteins and understanding metal-coupled protein folding mechanisms are aspects of high importance for the structure-to-function relationship. Mass spectrometry (MS) has brought a powerful adjunct perspective to structural biology, obtaining from metal-to-protein stoichiometry to quaternary structure information. Currently, the different experimental and/or instrumental setups usually require the use of multiple data analysis software, and in some cases, they lack some of the main data analysis steps (MS processing, scoring, identification). Here, we present a comprehensive data analysis pipeline that addresses charge-state deconvolution, statistical scoring, and mass assignment for native MS, bottom-up, and native top-down with emphasis on metal-protein complexes. We have evaluated all of the approaches using assemblies of increasing complexity, including free and chemically labeled proteins, from low- to high-resolution MS. In all cases, the results have been compared with common software and proved how MetaOdysseus outperformed them.
We focused on the application of mass spectrometry and electrochemical methods combined with a chemometric analysis for the characterization of partially metallothionein-3 species. The results showed decreased Cat1 and Cat2 signals for the Zn(II)-loaded MT3 species with respect to the metal-free protein, which might be explained by the arrangement of tetrahedral metal-thiolate coordination environments and the formation of metal clusters. Moreover, there was a decrease in the Cat1 and Cat2 signals, and a plateau was reached with 4-5 Zn(II) ions that corresponded to the formation of the C-terminal α-domain. Regarding the Zn7-xMT3 complexes, we observed three different electrochemical behaviours for the Zn1-2MT3, Zn3-6MT3 and Zn7MT3 species. The difference for Zn1-2MT3 might be explained by the formation of independent ZnS4 cores in this stage that differ with respect to the formation of ZnxCysy clusters with an increased Zn(II) loading. The binding of the third Zn(II) ion to MT3 resulted in high sample heterogeneity due the co-existence of Zn3-6MT3. Finally, the Zn7MT3 protein showed a third type of behaviour. The fact that there were no free Cys residues might explain this phenomenon. Thus, this research identifies the major proteins responsible for zinc buffering in the cell.
- MeSH
- apoproteiny chemie MeSH
- elektrochemie metody MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- proteiny nervové tkáně chemie MeSH
- zinek chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
