Mass Spectrometric Analysis of Protein Phosphorylation
Phosphorylation is one the most frequently occurring posttranslational modifications in proteins, playing an essential role in transferring signals from the outside to the inside of a cell and in regulating many diverse cellular processes such as growth, metabolism, proliferation, motility, and differentiation. It is estimated that up to one third of all proteins in a typical mammalian cell are phosphorylated (1). Phosphorylation is carried out by a vast group of protein kinases which are thought to constitute 3% of the entire eukaryotic genome (1-3). To decipher the recognition signal of protein kinases and protein phosphatases acting on a given molecular target, and to understand how the activity of the target protein is regulated by phosphorylation, it is important to define the sites and the extent of phosphorylation at each specific site.
KeywordsPhosphorylation Site Okadaic Acid Neutral Loss Calf Intestinal Alkaline Phosphatase Micropipet Puller
- 15.Watts, J. D., Affolter, M., Krebs, D. L., Wange, R. L., Samelson, L. E., and Aebersold, R. (1994) Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70. J. Biol. Chem. 269, 29,520–29,529.PubMedGoogle Scholar
- 17.Schneider, U., Mini, T., Jenö, P., Fisher, P. A., and Stuurman, N. (1999) Phosphorylation of the major Drosophila lamin in vivo: site identification during both M-phase (meiosis) and interphase by electrospray ionization tandem mass spectrometry. Biochemistry 38, 4620-4632.Google Scholar
- 20.Covey, T. R. (1995) in Methods in Molecular Biology, Vol. 61, (Chapman, J. R., ed.), Humana Press, Totowa, NJ, pp. 83–99.Google Scholar
- 21.Covey, T., Shushan, B., Bonner, R., Schröder, W., and Hucho, F., (1991) in Methods in Protein Sequence Analysis (Jörnvall, H., Hoog, J. O., and Gustavsson, A. M. eds.), Birkhäuser, Basel, pp. 249–256.Google Scholar