Proteomic analysis was carried with liquid chromatography-tandem mass spectrometry (LC-MS/MS) after enzymatic digestion of the proteins to digested peptides in the bottom-up proteomics. Especially, the enrichment of phosphorylated peptides enables to analyze phosphopeptides with LC-MS/MS in this decade. Titanium dioxide, immobilized metal affinity chromatography (IMAC), and other metal-based affinity chromatography were widely used to enrich phosphorylated peptides. [2-8] The cells were lysed by sonication and the proteins digested with trypsin followed by enrichment of the phosphorylated peptides with these affinity chromatography. In our laboratory, the lysate was applied on MassPREPTM (Waters, Ma), which is aluminum-based affinity chromatography, according to the manufacturer’s protocol. On the analysis of phosphopeptides in mass spectrometer, neutral loss, which is the prior dissociation of phosphates from the parent mass in collision-induced dissociation, is frequently observed. A pseudo MS3 scan, also referred to as “multiple activation,” enables the analysis of neutral loss peptides and enhances detection [9]. Combined with recent advances in mass spectrometry, these technological advances have allowed the identification of hundreds to thousands of phosphorylation sites in a proteome [10-17].