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ProPerDP: A Protein Persulfide Detection Protocol

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Vascular Effects of Hydrogen Sulfide

Part of the book series: Methods in Molecular Biology ((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.

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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

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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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Authors and Affiliations

  1. Department of Molecular Immunology and Toxicology, National Institute of Oncology, Budapest, Hungary

    Éva Dóka & Péter Nagy

  2. Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    Elias S. J. Arnér

  3. Department of Microbiology and Immunology, Montana State University, Bozeman, MT, USA

    Edward E. Schmidt

Authors
  1. Éva Dóka
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  2. Elias S. J. Arnér
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  3. Edward E. Schmidt
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  4. Péter Nagy
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Corresponding author

Correspondence to Péter Nagy .

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Editors and Affiliations

  1. Department of Pathophysiology, Medical University, Lublin, Poland

    Jerzy Bełtowski

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Dóka, É., Arnér, E.S.J., Schmidt, E.E., Nagy, P. (2019). ProPerDP: A Protein Persulfide Detection Protocol. In: Bełtowski, J. (eds) Vascular Effects of Hydrogen Sulfide. Methods in Molecular Biology, vol 2007. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9528-8_5

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  • DOI: https://doi.org/10.1007/978-1-4939-9528-8_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9527-1

  • Online ISBN: 978-1-4939-9528-8

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