Skip to main content
Log in

Nucleic Acids in Protein Samples Interfere with Phosphopeptide Identification by Immobilized-Metal-Ion Affinity Chromatography and Mass Spectrometry

  • RESEARCH
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Immobilized-metal-ion affinity chromatography (IMAC) is used extensively for phosphopeptide enrichment in phosphoproteomics. However, the effect of nucleic acids in protein samples on phosphopeptide enrichment by IMAC has not yet been well clarified. In this study, we demonstrate that IMAC beads possess a strong adsorption of nucleic acids, especially single-stranded or single-stranded-region-containing nucleic acids, leading to approximately 50% loss of phosphopeptides during the process of IMAC enrichment. Therefore, nucleic acids must be removed from protein samples prior to IMAC. Acetonitrile (ACN) precipitation, a simple and efficient procedure, was established to remove nucleic acids from the protein samples. We showed that ACN precipitation approximately doubled the phosphopeptide number identified by IMAC and mass spectrometry, indicating that nucleic acid removal significantly improves the identification of phosphopeptides.

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
Fig. 3

Similar content being viewed by others

Abbreviations

ACN:

Acetonitrile

IMAC:

Immobilized-metal-ion affinity chromatography

LC:

Liquid chromatography

MALDI-TOF-MS:

Matrix-assisted desorption/ionization time-of-flight mass spectrometry

MS/MS:

Tandem mass spectrum

TFA:

Trifluoroacetic acid

References

  1. Cohen, P. (2000). The regulation of protein function by multisite phosphorylation—a 25 year update. Trends in Biochemical Sciences, 25, 596–601. doi:10.1016/S0968-0004(00)01712-6.

    Article  CAS  Google Scholar 

  2. Mann, M., Ong, S. E., Gronborg, M., Steen, H., Jensen, O. N., & Pandey, A. (2002). Analysis of protein phosphorylation using mass spectrometry: Deciphering the phosphoproteome. Trends in Biotechnology, 20, 261–268. doi:10.1016/S0167-7799(02)01944-3.

    Article  CAS  Google Scholar 

  3. Kokubu, M., Ishihama, Y., Sato, T., Nagasu, T., & Oda, Y. (2005). Specificity of immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for protein phosphorylation analysis. Analytical Chemistry, 77, 5144–5154. doi:10.1021/ac050404f.

    Article  CAS  Google Scholar 

  4. Kalume, D. E., Molina, H., & Pandey, A. (2003). Tackling the phosphoproteome: Tools and strategies. Current Opinion in Chemical Biology, 7, 64–69. doi:10.1016/S1367-5931(02)00009-1.

    Article  CAS  Google Scholar 

  5. Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., Shabanowitz, J., et al. (2002). Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nature Biotechnology, 20, 301–305. doi:10.1038/nbt0302-301.

    Article  CAS  Google Scholar 

  6. Murphy, J. C., Jewell, D. L., White, K. I., Fox, G. E., & Willson, R. C. (2003). Nucleic acid separations utilizing immobilized metal affinity chromatography. Biotechnology Progress, 19, 982–986. doi:10.1021/bp025563o.

    Article  CAS  Google Scholar 

  7. Cano, T., Murphy, J. C., Fox, G. E., & Willson, R. C. (2005). Separation of genomic DNA from plasmid DNA by selective renaturation with immobilized metal affinity capture. Biotechnology Progress, 21, 1472–1477. doi:10.1021/bp050155g.

    Article  CAS  Google Scholar 

  8. Potty, A. S., Fu, J. Y., Balan, S., Haymore, B. L., Hill, D. J., Fox, G. E., et al. (2006). Neutral additives enhance the metal-chelate affinity adsorption of nucleic acids: Role of water activity. Journal of Chromatography A, 1115, 88–92. doi:10.1016/j.chroma.2006.02.077.

    Article  CAS  Google Scholar 

  9. Ishihama, Y., Wei, F.-Y., Aoshima, K., Sato, T., Kuromitsu, J., & Oda, Y. (2007). Enhancement of the efficiency of phosphoproteomic identification by removing phosphates after phosphopeptide enrichment. Journal of Proteome Research, 6, 1139–1144. doi:10.1021/pr060452w.

    Article  CAS  Google Scholar 

  10. Gobom, J., Nordhoff, E., Mirgorodskaya, E., Ekman, R., & Roepstorff, P. (1999). Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. Journal of Mass Spectrometry, 34, 105–116. doi:10.1002/(SICI)1096-9888(199902)34:2<105::AID-JMS768>3.0.CO;2-4.

    Article  CAS  Google Scholar 

  11. Larsen, M. R., Cordwell, S. J., & Roepstorff, P. (2002). Graphite powder as an alternative or supplement to reversed-phase material for desalting and concentration of peptide mixtures prior to matrix-assisted laser desorption/ionization-mass spectrometry. Proteomics, 2, 1277–1287. doi:10.1002/1615-9861(200209)2:9<1277::AID-PROT1277>3.0.CO;2-P.

    Article  CAS  Google Scholar 

  12. Nuhse, T. S., Stensballe, A., Jensen, O. N., & Peck, S. C. (2003). Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry. Molecular & Cellular Proteomics, 2, 1234–1243. doi:10.1074/mcp.T300006-MCP200.

    Article  Google Scholar 

  13. Feng, S., Pan, C., Jiang, X., Xu, S., Zhou, H., Ye, M., et al. (2007). Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis. Proteomics, 7, 351–360. doi:10.1002/pmic.200600045.

    Article  CAS  Google Scholar 

  14. Jin, W. H., Dai, J., Zhou, H., Xia, Q. C., Zou, H. F., & Zeng, R. (2004). Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry. Rapid Communications in Mass Spectrometry, 18, 2169–2176. doi:10.1002/rcm.1604.

    Article  CAS  Google Scholar 

Download references

Acknowledgment

This study was partially supported by Natural Science Foundation of China Grant (30623009 and 30721063) and State Key Basic Research Program of China Grant (2007CB507404).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dexian Zheng.

Electronic supplementary material

Below is the link to the electronic supplementary material.

PDF 1250 kb

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, Y., Luo, Y., Wu, S. et al. Nucleic Acids in Protein Samples Interfere with Phosphopeptide Identification by Immobilized-Metal-Ion Affinity Chromatography and Mass Spectrometry. Mol Biotechnol 43, 59–66 (2009). https://doi.org/10.1007/s12033-009-9176-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-009-9176-6

Keywords

Navigation