Lu, L., Gao, X., Li, Q., Huang, J., Liu, R. and Li, H. 2012. Comparative phosphoproteomics analysis of the effects of L-methionine on dairy cow mammary epithelial cells. Can. J. Anim. Sci. 92: 433-442. L-methionine is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 (signal transducer and activator of transcription 5) and mTOR (mammalian target of rapamycin) regulate milk protein synthesis. But a comprehensive understanding of transcriptional and posttranscriptional regulation of milk protein synthesis is lacking. In the current study, two-dimensional gel electrophoresis (2-DE)/MS-based proteomics analysis was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effects of L-methionine on DCMECs were analyzed by CASY (Counter Analyser System) technique, reversed phase high performance liquid chromatography. The results showed that rate of cell proliferation and expression of β-casein were increased in DCMECs treated with 0.6 mM L-methionine for 24 h. Five proteins for which expression was significantly increased in DCMECs were selected, and their expression changes were verified by quantitative real-time PCR and Western blot analysis. The five up-regulated expressed phosphoproteins included Staphylococcal nuclease domain-containing protein 1(SND1), Septin-6, Glycyl-tRNA synthetase (GARS), Twinfilin-1 and eukaryotic elongation factor1-beta (eEF1B). This study revealed that availability of L-methionine influences the levels of nuclear phosphorylated proteins of DCMECs which opens a new avenue for the study of the molecular mechanism linking to milk protein synthesis.