Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins

• DOI: 10.1021/acs.jproteome.4c00271
• Publication Date: 2024-07-12T07:14:06Z
• Authors: Manuel David Peri...; Alicja Orzeł; Sylwia Wu; Karolina Mosna; Perdita E. Barran; Artur Krężel
• Keywords: Metallothionein; Iodoacetamide; Proteome; Metalloprotein; Isobaric labeling

Within the intricate landscape of proteome, approximately 30% all proteins bind metal ions. This repertoire is even larger when considering different forms a protein, known as proteoforms. Here, we propose term "metalloforms" to refer structural or functional variations protein resulting from binding various hetero- homogeneous Using human Cu(I)/Zn(II)-metallothionein-3 representative model, developed chemical proteomics strategy simultaneously differentiate and map Zn(II) Cu(I) sites. In first labeling step, N-ethylmaleimide reacts with Cysteine (Cys), in dissociation ions while remains bound protein. second iodoacetamide utilized label Cu(I)-bound Cys residues. Native mass spectrometry (MS) was used determine metal/labeling stoichiometries, bottom-up/top-down MS Cys-labeled Next, methodology interrogate an isolated rabbit liver metallothionein fraction containing three metallothionein-2 isoforms multiple Cd(II)/Zn(II) metalloforms. The approach detailed this study thus holds potential decode metalloproteoform diversity within other proteins.