Phosphorylation sites mapping

F. Zappacosta, M.J. Huddleston, R.L. Karcher, V.I. Gelfand, S.A. Carr, R.S. Annan, Improved sensitivity for phosphopeptide mapping using capillary LC and micro-ESI-MS comparative phosphorylation site mapping from gel-derived proteins. Anal. Chem., 74 (2002) 3221. 

Loyet, K. M., Stults, J. T., and Amott, D., Mass spectrometric contributions to the practice of phosphorylation site mapping through 2003 A literature review. Molecular Cell Prvteomics, 4,235-245,2005. 

When 14-3-3s were first identified as phosphorylation dependent binding proteins (note that a selection of non-phosphorylated targets are known), target protein phosphorylation sites were mapped and it was immediately apparent that 14-3-3s bound preferentially to specific phosphorylation motifs. The advent of oriented... 

Neubauer, G., and Mann, M. (1999). Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry potentials and hmitations. Anal. Chem. 71, 235-242. 

Dual Phosphorylation Sites in MAP Kinase Family Members... 

Each of these pathways involves a kinase cascade resulting in the phosphorylation and activation of the MAP kinase family member. Each contains a dual phosphorylation site (TEY, TPY, or TGY) and the central residue in the motif characteristic of the class, as shown in Table 8.1. It is evident that cells are endowed with parallel signal-transduction pathways and that they may operate individually or in combination to initiate specific patterns of gene expression. Additionally, crosstalk between the pathways undoubtedly occurs. None of these pathways has a unique function it is more likely that the combination of pathways that are activated (or silenced) together with the... 

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. 

Cao P. and Stults J.T. (2000), Mapping the phosphorylation sites of proteins using on-line immobilized metal-ion affinity chromatography/capillary electrophore-sis/electrospray ionization multiple stage tandem mass spectrometry, Rapid Commun. Mass Spectrom. 14(17), 1600-1606. 

Schlosser, A., Vanselow, J.T. and Kramer, A. (2005) Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis. Analytical Chemistry, 77, 5243-5250. 

Feshchenko, E. A., W. Y. Langdon, and A. Y. Tsygankov. Fyn, Yes, and Syk phosphorylation sites in c-Cbl map to the same tyrosine residues that become phosphorylated in activated T cells. J Biol Chem. 273 8323-8331.1998. 

Phosphorylation on serine, threonine, and tyrosine residues is an extremely important modulator of protein function. Phosphorylation can be analyzed by mass spectrometry with enrichment of compounds of interest using immobilized metal affinity chromatography and chemical tagging techniques, detection of phosphopep-tides using mass mapping and precursor ion scans, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags (McLachlin and Chait 2001). 

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. 

There are 85 putative serine or threonine phosphorylation sites on the longest CNS tau isoform. Phosphorylation sites were characterized by phospho-dependent tau antibodies, phopho-peptide mapping, mass spectrometry, and NMR. Most of the phosphorylation sites surround the microtubule-binding domains in the proline-rich... 

Fig. 6.4 Receptor-regulated SMADs (R-SMADs) have two homology regions, one at the amino- and one at the cartXM termini, MHl and MH2. They are linked by a proline-rich linker region. The iinker region is highly variable in size and sequence. It participates in the formation of homo-oligomeric structures and contains phosphorylation sites for the MAP kinase. Phosphorylation by the MAP kinase has a negative effect, because it prevents nuclear translocation of the SMADs. R-SMADs interact through the MH2 domain with the activated receptor I and are phosphorylated at the C-terminal SS(V/M)S motif, where S is serine V, valine and M, methionine. Both, MHl and MH2 domains are essential for DNA binding and the recruitment of DNA-binding proteins.

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). 

Software predictions suggest a number of potential phosphorylation sites in the coronin 7 protein. The highest E value predictions are for potential MAP kinase phosphorylation sites at serine residue 442 and threonines 497 and 733, cdc2 sites at S-450 and S-775, cdk5 at S-437, PKC sites at S-7, S-465 and T-654 and Src sites at tyrosine residues 288 and 758. Not surprisingly, many serine and threonine phosphorylation predictions concentrate in the low complexity PST-enriched region. Although it remains to be elucidated which of the predicted sites are relevant in vivo, at least some of them have been experimentally demonstrated to be phosphorylated in vitro (see below). 

Conventional methods for the study of protein phosphorylation rely on radioactive labelling, 2D-GE protein mapping, and Edman degradation. Early studies in LC-MS characterization of protein phosphorylation involve MS-MS analysis of modified tryptic peptide to determine the phosphorylation site by complementary peptide mapping, e.g., [5-7]. In the LC-MS analysis of tryptic and V8-protease digests of a phosphorylated (ppl9) and nonphosphorylated (pl9) 19-kDa cytosolic protein, two sets of ions with a phosphate-characteristic mass difference of 80 Da were observed. Sequence analysis of the relevant peptides by MS-MS showed that phosphorylation occurs at Ser-25 and Ser-38 [7]. 

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