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

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

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