In order to evaluate ORM2 could activate which signaling pathway, phosphoproteomic analysis was performed. First, liver proteins were extracted from ob/ob mice injected with Ad-ORM2 and Ad-GFP using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Next, the phosphorylated peptides were enriched by immobilized metal affinity chromatograph and the fractions were pre-separated by reversed phase liquid chromatograph. Electron transfer dissociation was used to effectively retain the skeletal information and phosphorylation modification information of the peptide, and accurately determine the molecular mass of proteins or peptides. Finally, the phosphoproteomic analysis was performed by tandem mass spectrometry and database retrieval.