Advertisement

A modified protein persulfidation detection method

Abstract

As a gastransmitter, hydrogen sulfide (H2S) plays an important role in regulating plant growth and stress resistance. The proposed functional mechanism of H2S is persulfidation, a kind of posttranslational modification on free cysteine residues of proteins, which is also called S-sulfhydration previously. At present, biotin switch assay is an effective method for protein persulfidation level detection, while the protein sample need be precipitated and resuspended repeatedly according to the current protocol. Here, we reported a modified method which adapted from biotin switch assay and reduced the repetitive steps mentioned above. The total protein of plant or purified recombination protein from E. coli was directly loaded on the activated NC membrane. The free sulfhydryl of cysteine residues of protein on NC membrane could be blocked with MMTS reagent, while the persulfidation modified cysteine residues would be labeled by biotin-HPDP. After that, the persulfidation level of proteins was detected by immunoblotting with anti-biotin antibodies. This method was also applicable to the plant proteins, which were transferred to NC membrane after separated by native PAGE. This method reduces the repeated steps of precipitation and resuspension of protein samples, and by this it could reduce the loss and content error of protein. Besides, it is more sensitive and convenient than the previous biotin switch assay method.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 99

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2

Abbreviations

DL:

Dithiothreitol (DTT)

NC membrane:

Nitrocellulose filter membrane

MMTS:

S-Methyl methanethiosulfonate

Biotin-HPDP:

S-N-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio) propinamide

References

  1. Ángeles A, Antonio S, Cecilia G, Luis CR (2015) S-Sulfhydration: a cysteine posttranslational modification in plant systems. Plant Physiol 168(1):334–342

  2. Aroca A, Benito JM, Gotor C, Romero LC (2017) Persulfidation proteome reveals the regulation of protein function by hydrogen sulfide in diverse biological processes in Arabidopsis. J Exp Bot 68(17):4915–4927

  3. Birke H, Haas FH, De Kok LJ, Balk J, Wirtz M, Hell R (2012) Cysteine biosynthesis, in concert with a novel mechanism, contributes to sulfide detoxification in mitochondria of Arabidopsis thaliana. Biochem J 445(2):275–283

  4. Conte ML, Carroll KS (2013) The redox biochemistry of protein sulfenylation and sulfinylation. J Biol Chem 288(37):26480–26488

  5. Gao XH, Krokowski D, Guan BJ, Bederman I, Majumder M, Parisien M, Diatchenko L et al (2015) Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response. eLIFE 4:e10067

  6. Jaffrey SR, Snyder SH (2001) The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE 2001(86):pl1

  7. Kimura H (2015) Hydrogen sulfide and polysulfides as signaling molecules. Proc Jpn Acad Ser B 91(4):131–159

  8. Krishnan N, Fu CX, Pappin D, Tonks NK (2011) H2S-induced sulfhydration of the phosphatase PTP1B and its role in the endoplasmic reticulum stress response. Sci Signal 4(203):ra86

  9. Liu ZQ, Yan L, Wu Z, Mei C, Lu K, Yu YT, Liang S, Zhang XF, Wang XF, Zhang DP (2012) Cooperation of three WRKY-domain transcription factors WRKY18, WRKY40, and WRKY60 in repressing two ABA-responsive genes ABI4 and ABI5 in Arabidopsis. J Exp Bot 63(18):6371–6392

  10. Liu ZQ, Li YW, Cao CY, Liang S, Ma YS, Liu X, Pei YX (2019) The role of H2S in low temperature-induced cucurbitacin C increases in cucumber. Plant Mol Biol 99(6):535–544

  11. Longen S, Richter F, Köhler 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

  12. Lu CY, Kavalier A, Lukyanov E, Gross SS (2013) S-Sulfhydration/desulfhydration and S-nitrosylation/denitrosylation: a common paradigm for gasotransmitter signaling by H2S and NO. Methods 62(2):177–181

  13. 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):ra2

  14. Pei YX (2016) Gasotransmitter hydrogen sulfide in plants: stinking to high heaven, but refreshing to fine life. Chin J Biochem Mol Biol 32(07):721–733

  15. Riemenschneider A, Nikiforova V, Hoefgen R, De Kok LJ, Papenbrock J (2005) Impact of elevated H2S on metabolite levels, activity of enzymes and expression of genes involved in cysteine metabolism. Plant Physiol Biochem 43(5):473–483

  16. Romero LC, Aroca MÁ, Laureano-Marín AM, Moreno I, García I, Gotor C (2014) Cysteine and cysteine-related signaling pathways in Arabidopsis thaliana. Mol Plant 7(2):264–276

  17. Sen N, Paul BD, Gadalla MM, Mustafa AK, Sen T, Xu RS, Kim S, Snyder SH (2012) Hydrogen sulfide-linked sulfhydration of NF-kappaB mediates its antiapoptotic actions. Mol Cell 45(1):13–24

  18. Shi HT, Ye TT, Han N, Bian HW, Liu XD, Chan ZL (2015) Hydrogen sulfide regulates abiotic stress tolerance and biotic stress resistance in Arabidopsis. J Integr Plant Biol 57(7):628–640

  19. Zhang DH, Macinkovic I, Devarie-Baez NO, Pan J, Park CM, Carroll KS, Filipovic MR, Xian M (2014) Detection of protein S-sulfhydration by a tag-switch technique. Angew Chem Int Ed 53(2):575–581

  20. Zhang D, Du JB, Tang CS, Huang YQ, Jin HF (2017) H2S-induced sulfhydration: biological function and detection methodology. Front Pharmacol 8:608

Download references

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31671605 to Yanxi Pei).

Author information

Correspondence to Yanxi Pei.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by B. Zheng.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, Z.Q., Jia, Y.Q., Li, Y.W. et al. A modified protein persulfidation detection method. Acta Physiol Plant 42, 16 (2020). https://doi.org/10.1007/s11738-019-3006-7

Download citation

Keywords

  • Persulfidation detection
  • Hydrogen sulfide
  • Biotin switch
  • Immunoblotting