Skip to main content

Advertisement

Log in

Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimensional electrophoresis

  • Published:
BioMetals Aims and scope Submit manuscript

Abstract

Copper (Cu) is an essential micronutrient required for plant growth and development. However, excess Cu can inactivate and disturb protein structure as a result of unavoidable binding to proteins. To understand better the mechanisms involved in Cu toxicity and tolerance in plants, we developed a new immobilized metal affinity chromatography (IMAC) method for the separation and isolation of Cu-binding proteins extracted from roots of rice seedling exposed to excess Cu. In our method, IDA-Sepharose or EDDS-Sepharose column (referred as pre-chromatography) and Cu-IDA-Sepharose column (referred as Cu-IMAC) were connected in tandem. Namely, protein samples were pre-chromatographed with IDA-Sepharose column to removal metal ions, then protein solution was flowed into Cu-IMAC column for enriching Cu-binding proteins in vitro. Compared with the control (Cu-IMAC without any pre-chromatography), IDA-Sepharose pre-chromatography method markedly increased yield of the Cu-IMAC-binding proteins, and number of protein spots and the abundance of 40 protein spots on two-dimensional electrophoresis (2-DE) gels. Thirteen protein spots randomly selected from 2-DE gel and 11 proteins were identified using MALDI-TOF-TOF MS. These putative Cu-binding proteins included those involved in antioxidant defense, carbohydrate metabolism, nucleic acid metabolism, protein folding and stabilization, protein transport and cell wall synthesis. Ten proteins contained one or more of nine putative metal-binding motifs reported by Smith et al. (J Proteome Res 3:834–840, 2004) and seven proteins contained one or two of top six motifs reported by Kung et al. (Proteomics 6:2746–2758, 2006). Results demonstrated that more proteins specifically bound with Cu-IMAC could be enriched through removal of metal ions from samples by IDA-Sepharose pre-chromatography. Further studies are needed on metal-binding characteristics of these proteins in vivo and the relationship between Cu ions and protein biological activities to fully understand the mechanisms of Cu tolerance and toxicity in plants.

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

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

Download references

Acknowledgments

This work was financially supported by the Fundamental Research Funds for the Central Universities (KYZ200905 and KYZ201316), Specialized Research Fund for the Doctoral Program of Higher Education (20120097130004) and the National Natural Science Foundation of China (31172021).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Zhenguo Shen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Song, Y., Zhang, H., Chen, C. et al. Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimensional electrophoresis. Biometals 27, 265–276 (2014). https://doi.org/10.1007/s10534-014-9707-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10534-014-9707-x

Keywords

Navigation