|
KMID : 0370220080520050407
|
|
Yakhak Hoeji
2008 Volume.52 No. 5 p.407 ~ p.411
|
|
|
Optimization of TiO2 Method to Identify the Phosphorylation Sites of α-Casein
|
|
Kim Hye-Jeong
Park Ja-Hye
Baek Moon-Chang
|
|
|
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 (TiO2) method should be combined with MS/MS analysis to effectively identify phosphorylation sites. It is necessary to optimize phosphopeptide-enrichment strategy, TiO2 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 TiO2 method, we used ??-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 TiO2. This optimized method would assist in reliable identifying thousands of phosphorylation sites existed in low abundance from various complex proteins.
|
|
|
KEYWORD
|
|
|
TiO2 method, phosphorylation sites, α-casein
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
|