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ProPerDP: A Protein Persulfide Detection Protocol

  • Éva Dóka
  • Elias S. J. Arnér
  • Edward E. Schmidt
  • Péter NagyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2007)

Abstract

Persulfide or polysulfide formation on Cys residues is emerging as an abundant protein posttranslational modification, with important regulatory functions. However, as many other Cys oxidative modifications, per- and polysulfides are relatively labile, dynamically interchanging species, which makes their intracellular detections challenging. Here we report our recently developed highly selective method, Protein Persulfide Detection Protocol (ProPerDP), which can detect protein per- and polysulfide species in isolated protein systems, in blood plasma, or in cells and tissue samples. The method is easy to use and relatively inexpensive and requires only readily commercially available reagents. The biggest advantage of ProPerDP compared to other previously published persulfide detecting methods is the fact that in this protocol, all thiol and persulfide species are appropriately alkylated before any cell lysis step. This greatly reduces the potential of detecting lysis-induced oxidation-driven artifact persulfide formation.

Key words

Protein persulfide ProPerDP Detection method Biotin pulldown assay Selective reduction 

Abbreviations

A549

Adenocarcinomic human alveolar basal epithelial cells

ACN

Acetonitrile

BCA

Bicinchoninic acid

BCIP

5-Bromo-4-chloro-3′-indolyl phosphate p-toluidine salt

BSA

Bovine serum albumin

CHAPS

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate

Cys

Cysteine

DMEM-F12

Dulbecco’s modified eagle medium with F12 nutrient mixture

DTNB

5,5′-Dithiobis(2-nitrobenzoic acid), Ellman’s reagent

DTPA

Diethylenetriaminepentaacetic acid

DTT

Dithiothreitol

EDTA

Ethylenediaminetetraacetic acid

EGTA

Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

EMEM

Eagle’s minimum essential medium

FBS

Heat-inactivated fetal bovine serum

HBSS

Hank’s Balanced Salt Solution

HEK293

Human embryonic kidney cells 293

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HSA

Human serum albumin

IAB

EZ-Link™ Iodoacetyl-PEG2-Biotin

IAF

5-Iodoacetamido fluorescein

IAM

Iodoacetamide

NBT

Nitro-blue tetrazolium chloride

PBS

Phosphate-buffered saline

PIC

Protease inhibitor cocktail

Pipes

Piperazine-N,N′-bis(2-ethanesulfonic acid)

ProPerDP

Protein persulfide detection protocol

PVDF

Polyvinylidene fluoride

SB

SDS sample buffer, nonreducing, 4×

TCEP

Tris(2-carboxyethyl)phosphine

TE

100 mM Tris–HCl, 2 mM EDTA, pH = 7.4

TNB-

2-Nitro-5-thiobenzoate

TR/GR-null

Mouse liver lacking thioredoxin reductase and glutathione reductase

Trx

Thioredoxin

TTBS

20 mM Tris, 0.5 M NaCl, pH 7.5 + 0.05% Tween 20

Notes

Acknowledgments

Financial support from the Hungarian National Science Foundation (OTKA; grant no.: K109843, KH17_126766, and K18_129286) for P.N.; from the National Institutes of Health (grant no.: R21AG055022-01) for E.E.S., P.N., and E.S.J.A.; and from the Swedish Research Council, Swedish Cancer Society, and Karolinska Institutet for E.S.J.A. is acknowledged. P.N. is a János Bolyai Research Scholar of the Hungarian Academy of Sciences. Dojindo Molecular Technologies Inc. is greatly acknowledged for their kind support of chemical supplies.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Éva Dóka
    • 1
  • Elias S. J. Arnér
    • 2
  • Edward E. Schmidt
    • 3
  • Péter Nagy
    • 1
    Email author
  1. 1.Department of Molecular Immunology and ToxicologyNational Institute of OncologyBudapestHungary
  2. 2.Division of Biochemistry, Department of Medical Biochemistry and BiophysicsKarolinska InstitutetStockholmSweden
  3. 3.Department of Microbiology and ImmunologyMontana State UniversityBozemanUSA

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