Here, using human metallothionein (MT2) as an example, we describe an improved strategy based on differential alkylation coupled to MS, assisted by zinc probe monitoring, for identification of cysteine-rich binding sites with nanomolar and picomolar metal affinity utilizing iodoacetamide (IAM) and N-ethylmaleimide reagents. We concluded that an SN2 reaction provided by IAM is more suitable to label free Cys residues, avoiding nonspecific metal dissociation. Afterward, metal-bound Cys can be easily labeled in a nucleophilic addition reaction after separation by reverse-phase C18 at acidic pH. Finally, we evaluated the efficiency of the method by mapping metal-binding sites of Zn7-xMT species using a bottom-up MS approach with respect to metal-to-protein affinity and element(al) resolution. The methodology presented might be applied not only for MT2 but to identify metal-binding sites in other Cys-containing proteins.

Central European Institute of Technology Brno University of Technology Purkynova 123 612 00 Brno Czech Republic

Department of Chemical Biology Faculty of Biotechnology University of Wrocław F Joliot Curie 14a 50 383 Wrocław Poland

Department of Chemistry and Biochemistry Mendel University in Brno Zemedelska 1 613 00 Brno Czech Republic

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