Publication detail

Metal- and Affinity-Specific Dual Labeling of Cysteine-Rich Proteins for Identification of Metal-Binding Sites

PERIS-DÍAZ, M. GURÁŇ, R. ZÍTKA, O. ADAM, V. KRĘŻEL, A.

Original Title

Metal- and Affinity-Specific Dual Labeling of Cysteine-Rich Proteins for Identification of Metal-Binding Sites

Type

journal article in Web of Science

Language

English

Original Abstract

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 Nethylmaleimide 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.

Keywords

metalloprotein; chemical protein labeling; metallothionein; mass spectrometry

Authors

PERIS-DÍAZ, M.; GURÁŇ, R.; ZÍTKA, O.; ADAM, V.; KRĘŻEL, A.

Released

3. 8. 2020

Publisher

American Chemical Society

ISBN

0003-2700

Periodical

ANALYTICAL CHEMISTRY

Year of study

92

Number

19

State

United States of America

Pages from

12950

Pages to

12958

Pages count

9

URL

https://pubs.acs.org/doi/10.1021/acs.analchem.0c01604

Full text in the Digital Library

http://hdl.handle.net/11012/195633

BibTex

@article{BUT165899,
  author="Manuel David {Peris-Díaz} and Roman {Guráň} and Ondřej {Zítka} and Vojtěch {Adam} and Artur {Krężel}",
  title="Metal- and Affinity-Specific Dual Labeling of Cysteine-Rich Proteins for Identification of Metal-Binding Sites",
  journal="ANALYTICAL CHEMISTRY",
  year="2020",
  volume="92",
  number="19",
  pages="12950--12958",
  doi="10.1021/acs.analchem.0c01604",
  issn="0003-2700",
  url="https://pubs.acs.org/doi/10.1021/acs.analchem.0c01604"
}