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 ZnMT3 complexes, we observed three different electrochemical behaviours for the ZnMT3, ZnMT3 and ZnMT3 species. The difference for ZnMT3 might be explained by the formation of independent ZnS cores in this stage that differ with respect to the formation of ZnCys 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 ZnMT3. Finally, the ZnMT3 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.