A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags

B. Söveges, T. Imre, T. Szende, L. Póti, G. B. Cserép, T. Hegedűs, P. Kele, K. Németh

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Fluorescent tagging of proteins via accessible cysteine residues is of paramount importance. In this study, model proteins of interest (mitogen-activated protein kinases) were labeled successfully in native state on their free thiols by direct fluorescence derivatization, or in a sequential manner where conjugation of the site specific linker and the fluorophore is carried out in two steps. To this end we designed and prepared two novel chemical reporters carrying vinyl sulfone as Cys targeting function and cyclooctyne motifs, suitable for subsequent conjugation with fluorogenic azides via copper free strain-promoted azide-alkyne click chemistry. Direct and sequential labeling reaction steps were analyzed by native PAGE, capillary zone electrophoresis and tandem mass spectrometry. The efficiency of tagging was correlated with solvent accessibility of the Cys residues. Our results indicated that conjugation of native proteins by vinyl sulfone linkers was fast and thiol-selective. Subsequent click reaction with fluorogenic dyes generates intensive fluorescence signals and fulfills all requirements of bioorthogonality.

Original languageEnglish
Pages (from-to)6071-6078
Number of pages8
JournalOrganic and Biomolecular Chemistry
Volume14
Issue number25
DOIs
Publication statusPublished - 2016

Fingerprint

sulfones
cysteine
Labeling
marking
Cysteine
Azides
conjugation
proteins
Sulfhydryl Compounds
probes
Fluorescence
thiols
Click Chemistry
Native Polyacrylamide Gel Electrophoresis
Proteins
Alkynes
Fluorophores
Capillary Electrophoresis
Tandem Mass Spectrometry
Mitogen-Activated Protein Kinases

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Biochemistry

Cite this

A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags. / Söveges, B.; Imre, T.; Szende, T.; Póti, L.; Cserép, G. B.; Hegedűs, T.; Kele, P.; Németh, K.

In: Organic and Biomolecular Chemistry, Vol. 14, No. 25, 2016, p. 6071-6078.

Research output: Contribution to journalArticle

@article{8a8215ae4f334f8da9883b01ae4ebd97,
title = "A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags",
abstract = "Fluorescent tagging of proteins via accessible cysteine residues is of paramount importance. In this study, model proteins of interest (mitogen-activated protein kinases) were labeled successfully in native state on their free thiols by direct fluorescence derivatization, or in a sequential manner where conjugation of the site specific linker and the fluorophore is carried out in two steps. To this end we designed and prepared two novel chemical reporters carrying vinyl sulfone as Cys targeting function and cyclooctyne motifs, suitable for subsequent conjugation with fluorogenic azides via copper free strain-promoted azide-alkyne click chemistry. Direct and sequential labeling reaction steps were analyzed by native PAGE, capillary zone electrophoresis and tandem mass spectrometry. The efficiency of tagging was correlated with solvent accessibility of the Cys residues. Our results indicated that conjugation of native proteins by vinyl sulfone linkers was fast and thiol-selective. Subsequent click reaction with fluorogenic dyes generates intensive fluorescence signals and fulfills all requirements of bioorthogonality.",
author = "B. S{\"o}veges and T. Imre and T. Szende and L. P{\'o}ti and Cser{\'e}p, {G. B.} and T. Hegedűs and P. Kele and K. N{\'e}meth",
year = "2016",
doi = "10.1039/c6ob00810k",
language = "English",
volume = "14",
pages = "6071--6078",
journal = "Organic and Biomolecular Chemistry",
issn = "1477-0520",
publisher = "Royal Society of Chemistry",
number = "25",

}

TY - JOUR

T1 - A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags

AU - Söveges, B.

AU - Imre, T.

AU - Szende, T.

AU - Póti, L.

AU - Cserép, G. B.

AU - Hegedűs, T.

AU - Kele, P.

AU - Németh, K.

PY - 2016

Y1 - 2016

N2 - Fluorescent tagging of proteins via accessible cysteine residues is of paramount importance. In this study, model proteins of interest (mitogen-activated protein kinases) were labeled successfully in native state on their free thiols by direct fluorescence derivatization, or in a sequential manner where conjugation of the site specific linker and the fluorophore is carried out in two steps. To this end we designed and prepared two novel chemical reporters carrying vinyl sulfone as Cys targeting function and cyclooctyne motifs, suitable for subsequent conjugation with fluorogenic azides via copper free strain-promoted azide-alkyne click chemistry. Direct and sequential labeling reaction steps were analyzed by native PAGE, capillary zone electrophoresis and tandem mass spectrometry. The efficiency of tagging was correlated with solvent accessibility of the Cys residues. Our results indicated that conjugation of native proteins by vinyl sulfone linkers was fast and thiol-selective. Subsequent click reaction with fluorogenic dyes generates intensive fluorescence signals and fulfills all requirements of bioorthogonality.

AB - Fluorescent tagging of proteins via accessible cysteine residues is of paramount importance. In this study, model proteins of interest (mitogen-activated protein kinases) were labeled successfully in native state on their free thiols by direct fluorescence derivatization, or in a sequential manner where conjugation of the site specific linker and the fluorophore is carried out in two steps. To this end we designed and prepared two novel chemical reporters carrying vinyl sulfone as Cys targeting function and cyclooctyne motifs, suitable for subsequent conjugation with fluorogenic azides via copper free strain-promoted azide-alkyne click chemistry. Direct and sequential labeling reaction steps were analyzed by native PAGE, capillary zone electrophoresis and tandem mass spectrometry. The efficiency of tagging was correlated with solvent accessibility of the Cys residues. Our results indicated that conjugation of native proteins by vinyl sulfone linkers was fast and thiol-selective. Subsequent click reaction with fluorogenic dyes generates intensive fluorescence signals and fulfills all requirements of bioorthogonality.

UR - http://www.scopus.com/inward/record.url?scp=84975885821&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84975885821&partnerID=8YFLogxK

U2 - 10.1039/c6ob00810k

DO - 10.1039/c6ob00810k

M3 - Article

VL - 14

SP - 6071

EP - 6078

JO - Organic and Biomolecular Chemistry

JF - Organic and Biomolecular Chemistry

SN - 1477-0520

IS - 25

ER -