ProPerDP: A Protein Persulfide Detection Protocol

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


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 



Adenocarcinomic human alveolar basal epithelial cells




Bicinchoninic acid


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


Bovine serum albumin


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




Dulbecco’s modified eagle medium with F12 nutrient mixture


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


Diethylenetriaminepentaacetic acid




Ethylenediaminetetraacetic acid


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


Eagle’s minimum essential medium


Heat-inactivated fetal bovine serum


Hank’s Balanced Salt Solution


Human embryonic kidney cells 293


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


Human serum albumin


EZ-Link™ Iodoacetyl-PEG2-Biotin


5-Iodoacetamido fluorescein




Nitro-blue tetrazolium chloride


Phosphate-buffered saline


Protease inhibitor cocktail


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


Protein persulfide detection protocol


Polyvinylidene fluoride


SDS sample buffer, nonreducing, 4×




100 mM Tris–HCl, 2 mM EDTA, pH = 7.4




Mouse liver lacking thioredoxin reductase and glutathione reductase




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



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.


  1. 1.
    Doka E, Pader I, Biro A, Johansson K, Cheng Q, Ballago K, Prigge JR, Pastor-Flores D, Dick TP, Schmidt EE, Arner ES, Nagy P (2016) A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems. Sci Adv 2(1):e1500968. Scholar
  2. 2.
    Mustafa AK, Gadalla MM, Sen N, Kim S, Mu WT, Gazi SK, Barrow RK, Yang GD, Wang R, Snyder SH (2009) H2S signals through protein S-sulfhydration. Sci Signal 2(96):ARTN ra72. Scholar
  3. 3.
    Nagy P (2015) Mechanistic chemical perspective of hydrogen sulfide signaling. Methods Enzymol 554:3–29. Scholar
  4. 4.
    Ida T, Sawa T, Ihara H, Tsuchiya Y, Watanabe Y, Kumagai Y, Suematsu M, Motohashi H, Fujii S, Matsunaga T, Yamamoto M, Ono K, Devarie-Baez NO, Xian M, Fukuto JM, Akaike T (2014) Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling. Proc Natl Acad Sci USA 111(21):7606–7611. Scholar
  5. 5.
    Cuevasanta E, Moller MN, Alvarez B (2016) Biological chemistry of hydrogen sulfide and persulfides. Arch Biochem Biophys. Scholar
  6. 6.
    Yadav PK, Martinov M, Vitvitsky V, Seravalli J, Wedmann R, Filipovic MR, Banerjee R (2016) Biosynthesis and reactivity of cysteine persulfides in signaling. J Am Chem Soc 138(1):289–299. Scholar
  7. 7.
    Millikin R, Bianco CL, White C, Saund SS, Henriquez S, Sosa V, Akaike T, Kumagai Y, Soeda S, Toscano JP, Lin J, Fukuto JM (2016) The chemical biology of protein hydropersulfides: studies of a possible protective function of biological hydropersulfide generation. Free Radic Biol Med 97:136–147. Scholar
  8. 8.
    Bianco CL, Chavez TA, Sosa V, Saund SS, Nguyen QN, Tantillo DJ, Ichimura AS, Toscano JP, Fukuto JM (2016) The chemical biology of the persulfide (RSSH)/perthiyl (RSS.) redox couple and possible role in biological redox signaling. Free Radic Biol Med 101:20–31. Scholar
  9. 9.
    Bailey TS, Pluth MD (2015) Reactions of isolated persulfides provide insights into the interplay between H2S and persulfide reactivity. Free Radic Biol Med 89:662–667. Scholar
  10. 10.
    Ono K, Akaike T, Sawa T, Kumagai Y, Wink DA, Tantillo DJ, Hobbs AJ, Nagy P, Xian M, Lin J, Fukuto JM (2014) Redox chemistry and chemical biology of H2S, hydropersulfides, and derived species: implications of their possible biological activity and utility. Free Radic Biol Med 77:82–94. Scholar
  11. 11.
    Jung M, Kasamatsu S, Matsunaga T, Akashi S, Ono K, Nishimura A, Morita M, Abdul Hamid H, Fujii S, Kitamura H, Sawa T, Ida T, Motohashi H, Akaike T (2016) Protein polysulfidation-dependent persulfide dioxygenase activity of ethylmalonic encephalopathy protein 1. Biochem Biophys Res Commun 480(2):180–186. Scholar
  12. 12.
    Paul BD, Snyder SH (2012) H2S signalling through protein sulfhydration and beyond. Nat Rev Mol Cell Biol 13(8):499–507. Scholar
  13. 13.
    Wedmann R, Onderka C, Wei S, Szijarto IA, Miljkovic JL, Mitrovic A, Lange M, Savitsky S, Yadav PK, Torregrossa R, Harrer EG, Harrer T, Ishii I, Gollasch M, Wood ME, Galardon E, Xian M, Whiteman M, Banerjee R, Filipovic MR (2016) Improved tag-switch method reveals that thioredoxin acts as depersulfidase and controls the intracellular levels of protein persulfidation. Chem Sci 7(5):3414–3426. Scholar
  14. 14.
    Greiner R, Palinkas Z, Basell K, Becher D, Antelmann H, Nagy P, Dick TP (2013) Polysulfides link H2S to protein thiol oxidation. Antioxid Redox Signaling 19(15):1749–1765. Scholar
  15. 15.
    Nagy P, Winterbourn CC (2010) Rapid reaction of hydrogen sulfide with the neutrophil oxidant hypochlorous acid to generate polysulfides. Chem Res Toxicol 23(10):1541–1543. Scholar
  16. 16.
    Nagy P, Palinkas Z, Nagy A, Budai B, Toth I, Vasas A (2014) Chemical aspects of hydrogen sulfide measurements in physiological samples. Biochim Biophys Acta 1840(2):876–891. Scholar
  17. 17.
    Vasas A, Doka E, Fabian I, Nagy P (2015) Kinetic and thermodynamic studies on the disulfide-bond reducing potential of hydrogen sulfide. Nitric Oxide 46:93–101. Scholar
  18. 18.
    Shen X, Peter EA, Bir S, Wang R, Kevil CG (2012) Analytical measurement of discrete hydrogen sulfide pools in biological specimens. Free Radic Biol Med 52(11–12):2276–2283. Scholar
  19. 19.
    Wintner EA, Deckwerth TL, Langston W, Bengtsson A, Leviten D, Hill P, Insko MA, Dumpit R, VandenEkart E, Toombs CF, Szabo C (2010) A monobromobimane-based assay to measure the pharmacokinetic profile of reactive sulphide species in blood. Br J Pharmacol 160(4):941–957. Scholar
  20. 20.
    Rabilloud T, Vuillard L, Gilly C, Lawrence JJ (1994) Silver-staining of proteins in polyacrylamide gels: a general overview. Cell Mol Biol 40(1):57–75PubMedGoogle Scholar
  21. 21.
    Havlis J, Thomas H, Sebela M, Shevchenko A (2003) Fast-response proteomics by accelerated in-gel digestion of proteins. Anal Chem 75(6):1300–1306CrossRefGoogle Scholar
  22. 22.
    Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68(5):850–858CrossRefGoogle Scholar
  23. 23.
    Eriksson S, Prigge JR, Talago EA, Arner ES, Schmidt EE (2015) Dietary methionine can sustain cytosolic redox homeostasis in the mouse liver. Nat Commun 6:6479. Scholar
  24. 24.
    Longen S, Richter F, Kohler Y, Wittig I, Beck KF, Pfeilschifter J (2016) Quantitative persulfide site identification (qPerS-SID) reveals protein targets of H2S releasing donors in mammalian cells. Sci Rep 6:29808. Scholar
  25. 25.
    Alvarez B, Carballal S, Turell L, Radi R (2010) Formation and reactions of sulfenic acid in human serum albumin. Methods Enzymol 473:117–136. Scholar
  26. 26.
    Miseta A, Csutora P (2000) Relationship between the occurrence of cysteine in proteins and the complexity of organisms. Mol Biol Evol 17(8):1232–1239CrossRefGoogle Scholar
  27. 27.
    Holmberg A, Blomstergren A, Nord O, Lukacs M, Lundeberg J, Uhlen M (2005) The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures. Electrophoresis 26(3):501–510. Scholar

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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