Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins.
J Proteome Res
; 23(8): 3626-3637, 2024 Aug 02.
Article
em En
| MEDLINE
| ID: mdl-38993068
ABSTRACT
Within the intricate landscape of the proteome, approximately 30% of all proteins bind metal ions. This repertoire is even larger when considering all the different forms of a protein, known as proteoforms. Here, we propose the term "metalloforms" to refer to different structural or functional variations of a protein resulting from the binding of various hetero- or homogeneous metal ions. Using human Cu(I)/Zn(II)-metallothionein-3 as a representative model, we developed a chemical proteomics strategy to simultaneously differentiate and map Zn(II) and Cu(I) metal binding sites. In the first labeling step, N-ethylmaleimide reacts with Cysteine (Cys), resulting in the dissociation of all Zn(II) ions while Cu(I) remains bound to the protein. In the second labeling step, iodoacetamide is utilized to label Cu(I)-bound Cys residues. Native mass spectrometry (MS) was used to determine the metal/labeling protein stoichiometries, while bottom-up/top-down MS was used to map the Cys-labeled residues. Next, we used a developed methodology to interrogate an isolated rabbit liver metallothionein fraction containing three metallothionein-2 isoforms and multiple Cd(II)/Zn(II) metalloforms. The approach detailed in this study thus holds the potential to decode the metalloproteoform diversity within other proteins.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
/
Zinco
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Cobre
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Proteômica
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Metalotioneína
Limite:
Animals
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Humans
Idioma:
En
Revista:
J Proteome Res
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Polônia