YAKHAK HOEJI (약학회지)

The Pharmaceutical Society of Korea (대한약학회)
권호 표지

Optimization of $TiO_2$ Method to Identify the Phosphorylation Sites of ${\apha}$-Casein

${\apha}$-Casein의 인산화 위치 규명을 위한 티타늄 다이옥사이드($TiO_2$) 방법의 최적화

  • Kim, Hye-Jeong (Department of Molecular Medicine, School of Medicine, Kyungpook National University) ;
  • Park, Ja-Hye (Department of Molecular Medicine, School of Medicine, Kyungpook National University) ;
  • Baek, Moon-Chang (Department of Molecular Medicine, School of Medicine, Kyungpook National University)
  • 김혜정 (경북대학교 의과대학 분자의학교실) ;
  • 박자혜 (경북대학교 의과대학 분자의학교실) ;
  • 백문창 (경북대학교 의과대학 분자의학교실)
  • Published : 2008.10.31
Download PDF
⟨ Previous Next ⟩

Abstract

Phosphorylation plays the most important role in cell signaling mechanism. Various methods to identify the phosphorylation sites of proteins using tandem mass spectrometry (MS/MS) have been reported recently. Furthermore, the enrichment strategy such as Titanium dioxide ($TiO_2$) method should be combined with MS/MS analysis to effectively identify phosphorylation sites. It is necessary to optimize phosphopeptide-enrichment strategy, $TiO_2$ method in this study, due to the low amount of phosphorylated form followed by analyzing them by MS/MS. To evaluate the several conditions to enrich phosphopeptides using $TiO_2$ method, we used ${\apha}$-casein as a standard phosphoprotein and analyzed a representative phosphopeptide (VPQLEIVPNpSAEER) peak of MS spectrum. Batch is better than column method for binding and 300 g/l DHB in loading buffer is better than lower concentration of DHB. 3% TFA and pH 10.5 shows high efficiency of phosphopeptide-enrichment for washing and elution steps, respectively. Finally we identified various efficient conditions of phosphopeptide-enrichment method using $TiO_2$. This optimized method would assist in reliable identifying thousands of phosphorylation sites existed in low abundance from various complex proteins.

Keywords

References

  1. Hunter, T. : The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine: its role in cell growth and disease. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 353, 583 (1998) https://doi.org/10.1098/rstb.1998.0228
  2. Hunter, T. : Signaling 2000 and beyond. Cell 100, 113 (2000) https://doi.org/10.1016/S0092-8674(00)81688-8
  3. Pawson, T. and Nash, P. : Assembly of cell regulatory systems through protein interaction domains. Science 300, 445 (2003) https://doi.org/10.1126/science.1083653
  4. Baek, M. C., Krosky, P. M., He, Z. and Coen, D. M. : Specific phosphorylation of exogenous protein and peptide substrates by the human cytomegalovirus UL97 protein kinase. Importance of the P+5 position. J. Biol. Chem. 277, 29593 (2002) https://doi.org/10.1074/jbc.M202312200
  5. Aebersold, R. and Mann, M. : Mass spectrometry-based proteomics. Nature 422, 198 (2003) https://doi.org/10.1038/nature01511
  6. Chen, W. G. and White, F. M. : Proteomic analysis of cellular signaling. Expert. Rev. Proteomics 1, 343 (2004) https://doi.org/10.1586/14789450.1.3.343
  7. Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., Shabanowitz, J., Hunt, D. F. and White, F. M. : Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301 (2002) https://doi.org/10.1038/nbt0302-301
  8. Mumby, M. and Brekken, D. : Phosphoproteomics: new insights into cellular signaling. Genome Biol. 6, 230 (2005) https://doi.org/10.1186/gb-2005-6-9-230
  9. Rush, J., Moritz, A., Lee, K. A., Guo, A., Goss, V. L., Spek, E. J., Zhang, H., Zha, X. M., Polakiewicz, R. D. and Comb, M. J. : Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol. 23, 94 (2005) https://doi.org/10.1038/nbt1046
  10. Olsen, J. V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P. and Mann, M. : Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635 (2006) https://doi.org/10.1016/j.cell.2006.09.026
  11. Villen, J., Beausoleil, S. A., Gerber, S. A. and Gygi, S. P. : Large-scale phosphorylation analysis of mouse liver. Proc. Natl. Acad. Sci. U.S.A. 104, 1488 (2007)
  12. Wilson-Grady, J. T., Villen, J. and Gygi, S. P. : Phosphoproteome analysis of fission yeast. J. Proteome Res. 7, 1088 (2008) https://doi.org/10.1021/pr7006335
  13. Pinkse, M. W., Uitto, P. M., Hilhorst, M. J., Ooms, B. and Heck, A. J. : Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLCESI- MS/MS and titanium oxide precolumns. Anal. Chem. 76, 3935 (2004) https://doi.org/10.1021/ac0498617
  14. Larsen, M. R., Thingholm, T. E., Jensen, O. N., Roepstorff, P., and Jorgensen, T. J. : Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 4, 873 (2005) https://doi.org/10.1074/mcp.T500007-MCP200