Detail publikace

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.

Originální název

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

Typ

článek v časopise ve Web of Science, Jimp

Jazyk

angličtina

Originální abstrakt

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.

Klíčová slova

metalloprotein; chemical protein labeling; metallothionein; mass spectrometry

Autoři

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

Vydáno

3. 8. 2020

Nakladatel

American Chemical Society

ISSN

0003-2700

Periodikum

ANALYTICAL CHEMISTRY

Ročník

92

Číslo

19

Stát

Spojené státy americké

Strany od

12950

Strany do

12958

Strany počet

9

URL

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

Plný text v Digitální knihovně

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"
}