We performed here MS-based phosphoproteomics using both metal oxide affinity chromatography (pSTY proteomics) and anti-phosphotyrosine antibody (pY proteomics). The former method identified mainly phospho-serine and -threonine of nuclear or cytoplasmic proteins, whereas the latter did phosphotyrosine including more plasma membrane proteins and kinases. The overlap between these two methods was limited (24 tyrosine phosphorylation sites out of 325) and, by combining the two, coverage of the signaling molecules was enhanced as exemplified by Erk signaling. We also performed whole cell proteomics using an off-gel fractionator, and found 68.9% of the proteins identified by phosphoproteomics. Thus, the expression levels of phosphoproteins were roughly estimated. In addition to many uncharacterized phosphorylation sites, the dataset includes 136 sites that were experimentally verified elsewhere to be phosphorylated by a total of 83 kinases and kinase groups out of the 256 registered in the Phospho.ELM database. With the integration of various proteomic analyses and information from database, the responsible kinases of the identified phosphorylation sites and possibly their activity status were predicted by phosphorylation status and expression levels of their substrates, and thus our method may be able to monitor the activity status of phosphorylation signaling. (C) 2010 Elsevier B.V. All rights reserved.