α-Casein의 인산화 위치 규명을 위한 티타늄 다이옥사이드(TiO2) 방법의 최적화

Optimization of TiO2 Method to Identify the Phosphorylation Sites of α-Casein

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 α-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.