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

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dc.contributor.authorPeris-Díaz, Manuel Davidcs
dc.contributor.authorGuráň, Romancs
dc.contributor.authorZítka, Ondřejcs
dc.contributor.authorAdam, Vojtěchcs
dc.contributor.authorKrężel, Arturcs
dc.coverage.issue19cs
dc.coverage.volume92cs
dc.date.issued2020-08-03cs
dc.description.abstractHere, 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.en
dc.formattextcs
dc.format.extent12950-12958cs
dc.format.mimetypeapplication/pdfcs
dc.identifier.citationANALYTICAL CHEMISTRY. 2020, vol. 92, issue 19, p. 12950-12958.en
dc.identifier.doi10.1021/acs.analchem.0c01604cs
dc.identifier.issn0003-2700cs
dc.identifier.orcid0000-0002-2912-714Xcs
dc.identifier.orcid0000-0001-7607-5058cs
dc.identifier.orcid0000-0002-8527-286Xcs
dc.identifier.other165899cs
dc.identifier.researcheridC-2610-2016cs
dc.identifier.researcheridE11072012cs
dc.identifier.researcheridD-7686-2012cs
dc.identifier.scopus14012648400cs
dc.identifier.urihttp://hdl.handle.net/11012/195633
dc.language.isoencs
dc.publisherAmerican Chemical Societycs
dc.relation.ispartofANALYTICAL CHEMISTRYcs
dc.relation.urihttps://pubs.acs.org/doi/10.1021/acs.analchem.0c01604cs
dc.rightsCreative Commons Attribution 4.0 Internationalcs
dc.rights.accessopenAccesscs
dc.rights.sherpahttp://www.sherpa.ac.uk/romeo/issn/0003-2700/cs
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/cs
dc.subjectmetalloproteinen
dc.subjectchemical protein labelingen
dc.subjectmetallothioneinen
dc.subjectmass spectrometryen
dc.titleMetal- and Affinity-Specific Dual Labeling of Cysteine-Rich Proteins for Identification of Metal-Binding Sitesen
dc.type.driverarticleen
dc.type.statusPeer-revieweden
dc.type.versionpublishedVersionen
sync.item.dbidVAV-165899en
sync.item.dbtypeVAVen
sync.item.insts2025.02.03 15:50:00en
sync.item.modts2025.01.17 15:20:24en
thesis.grantorVysoké učení technické v Brně. Středoevropský technologický institut VUT. Chytré nanonástrojecs
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