Phosphorylation sites identification

Mass spectrometry of phosphopeptides has become a powerful tool for phosphorylation site identification. However, proteolytic digests examined by MS are often likely to fail to detect phosphopeptides because the ionization of phosphorylated peptides in positive ion mode MS is generally less efficient compared with the ionization of their nonphosphorylated counterparts resulting in ion suppression effects. A further problem is that phosphopeptides may not be retained by RP chromatography because they are too small and/or hydrophilic to bind to the CIS stationary phase. Therefore, capillary electrophoresis coupled to MS is a powerful method to enhance the detection of phosphoproteins and phosphopeptides due to their efficient separation by CE. 

Hogan, J.M. Pitteri, S.J. McLuckey, S.A. Phosphorylation site identification via ion trap tandem mass spectrometry of whole protein and peptide ions Bovine alpha-crystaUin A... 

Gillece-Castro, B. L. Arnott, D. P. Bier, M. E. Land, A. P. Stults, J. T. Peptide phosphorylation site identification A new method based on ion trap mass spectrometry. In Proceedings of the 43rd Conference on Mass Spectrometry and Allied Topics, Atlanta, GA, May 21-26,19 >S, p. 302. 

Krek, W and Nigg, E. A. (1991a). Differential phosphorylation of vertebrate p34c 2 kinase at the Gl/S and G2/M transtitions of the cell cycle identification of major phosphorylation sites. EMBO J. 10 305-316. 

Wang, X., Paulin, F. E., Campbell, L. E., Gomez, E., O Brien, K., Morrice, N., and Proud, C. G. (2001). Eukaryotic initiation factor 2B Identification of multiple phosphorylation sites in the epsilon-subunit and their functions in vivo. EMBO J. 20,... 

Filamentous tau is hyperphosphorylated. This is an early event that appears to precede filament assembly. It also renders tau unable to interact with microtubules. Much effort has gone into the mapping of phosphorylation sites and the identification of candidate protein kinases and phosphatases. For sites that are also phos-phorylated in normal brain tau, a higher proportion of tau molecules is phosphorylated in filamentous tau. In addition, filamentous tau is phosphorylated at more serine and threonine residues than tau from normal adult brain. Phosphorylation-dependent anti-tau antibodies were instrumental for the study of many phosphorylation sites. In particular, phosphorylation of S214 and S422 was found to be specific for assembled tau. 

Busman M., Schey K.L., Oatis Jr. J.E., and Knapp D.R.J. (1996), Identification of phosphorylation sites in phosphopeptides by positive and negative mode electrospray ionization tandem mass spectrometry, J. Am. Soc. Mass Spectrom. 7, 243-249. 

Fredericks, Z. L., Pitcher, J. A., and Lefkowitz, R. J. (1996) Identification of the G protein-coupled receptor kinase phosphorylation sites in the human beta2-adrenergic receptor. J. Biol. Chem. 271, 13796-13803. 

Guo, J., Wu, Y., Zhang, W., Zhao, L, Devi, L. A., Pei, G., and Ma, L. (2000) Identification of G protein-coupled receptor kinase 2 phosphorylation sites responsible for agonist-stimn-lated delta-opioid receptor phosphorylation. Mol. Pharmacol. 58, 1050-1056. 

The members of the protein kinase C family are central signal proteins and as such, are involved in the regulation of a multitude of cellular processes. A problem in the identification of substrates of protein kinase C is its low substrate specificity which often cannot be differentiated from that of protein kinase A, particularly in in vitro experiments. The consensus sequence of the phosphorylation sites in substrate proteins are similar to those of protein kinase A, in that basic amino acids are required in the neighborhood of the Ser/Thr residue to be phosphorylated. The following consensus sequences may be formulated for phosphorylation by protein kinase C ( = phosphorylation site) S /T XK/R K/RXXS /T K/RXXS /T XK/R K/RXS /T K/RXS /T XK/R (Pearson and Kemp, 1991). 

Munday, M.R. Campbell, D.G. Carling, D. Hardie, D.G. Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl-CoA carboxylase. Eur. J. Biochem., 175, 331-338 (1988)... 

Cote, G.P. Collins, J.H. Korn, E.D. Identification of three phosphorylation sites on each heavy chain of Acanthamoeba myosin II. J. Biol. Chem., 256, 12811-12816 (1981)... 

Williamson, B. L., Marchese, J., and Morrice, N. A. (2006). Automated identification and quantification of protein phosphorylation sites by LC/MS on a hybrid triple quadrupole linear ion trap mass spectrometer. Mol. Cell. Proteomics 5 337-346. 

D. R. Goodlett, Mass spectrometry-based methods for protein identification and phosphorylation site analysis, in ... 

Obermann, W. M., Gautel, M., Weber, K., and Furst, D. O. (1997). Molecular structure of the sarcomeric M band Mapping of titin and myosin binding domains in myomesin and the identification of a potential regulatory phosphorylation site in myomesin. EMBOJ. 16, 211-220. 

Orr GA, Han EK, Browne PC, et al. Identification of the major phosphorylation domain of murine mdrlb P-glycoprotein. Analysis of the protein kinase A and protein kinase C phosphorylation sites. J Biol Chem 1993 268(33) 25054-25062. 

Woods, A., Vertommen, D., Neumann, D., Turk, R., Bayliss, J., Schlattner, U., Wallimann, T., Carling, D., and Rider, M. H. 2003b. Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis. J Biol Chem 278 28434-28442. 

Rossie S, Gordon D, Catterall WA (1987) Identification of an intracellular domain of the sodium channel having multiple cAMP-dependent phosphorylation sites. J Biol Chem 252 17530-17535. 

Z3. Zhou, Z. X., Kempainen, J. A., and Wilson, E. M., Identification of three proline-directed phosphorylation sites in the human androgen receptor. Mol. Endocrinol. 9, 605-605 (1995). 

MALDI quadrupole ion trap mass spectrometry has also been used to localize and identify the post-translational modifications on the Sindai virus [18]. The polymerase associated protein (P protein) from this virus is reported in the literature to be highly phosphorylated. In vitro studies have detected phosphorylation in different regions of the protein, while a single phosphorylation site was found in the in vivo studies. Mass spectral data, along with computer-aided analysis, enabled the identification and localization of two phosphorylation sites. 

Douglas, P., Morrice, N., and MacKintosh, C., 1995, Identification of a regulatory phosphorylation site in the hinge 1 region of nitrate reductase from spinach (Spinacea oleracea) leaves, FEES Lett. 177 11311117. 

Johnson, M.K. (1969). The delayed neurotoxic effect of some organophosphorus compounds. Identification of the phosphorylation site as an esterase. Biochem. J. 114 711-17. 

Fractionation of proteins by strong cation exchange (SCX) chromatography, followed by IMAC enrichment of phosphopeptides from SCX fractions, led to a comprehensive identification of phosphoproteins of PSD isolated from mouse brain using LC-MS/MS (Trinidad et al. 2006). In this study, phosphorylation site(s) were mapped to 287 proteins from a total of 1,264 unique proteins identified. This translates into a 23% phosphorylation rate, comparable to an expected 33% rate in the general proteome (Johnson et al. 2005). The 287 phosphoproteins were derived from a total of 998 unique phosphorylated peptides, and the phosphorylations were mapped to 723 unique sites. Most of these occurred on serines, to a lesser extent on threonines, and only minimally on tyrosines (Figure 5A). 

Trinidad, J.C., Specht, C.G., Thalhammer, A., Schoepfer, R. and Burlingame, A.L. (2006) Comprehensive Identification of Phosphorylation Sites in Postsynaptic Density Preparations. Mol. Cell Proteomics 5, 914-922. 

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