Recent advances in methods for protein identification by a combination of mass spectrometry and protein/DNA sequence database enabled to study of high throughput analysis of proteome. In addition to expression level, numerous characterics of the proteins such as cellular localization, complex formation, stability, and post-transcriptional modifications can be studied by proteomic approach. However, whole proteome analysis has limitation to identify proteins with low expression level. Here we present that our strategy for purification of proteins with post-translational modifications, such as ubiquitylation and phosphorylation by affinity chromatography. Using antibodies against these modification groups, modified proteins were effectively purified. After digestion by trypsin, resulting peptides were subjected to online LC-ESI-MS/MS analysis. In addition to known substrate proteins, novel substrate proteins were identified. Thus, affinity purification of proteins that have post. translational modifications is effective method for focused proteomics.