Phosphopeptides

Lipase from Aspergillus niger, 0.2 M phosphate buffer, acetone, pH 7, 37°, 50-96% yield. This lipase was used in the cleavage of phosphopeptide heptyl esters. These conditions are sufficiently mild to prevent the elimination of phosphorylated serine and threonine residues." ... 

Scaffolding. Because 14-3-3s can bind potentially bind more than one phosphoprotein at once due the presence of two phosphopeptide-binding sites in a... 

It is not clear why some organisms have two 14-3-3 isoforms while others have up to 12. Binding 14-3-3 inhibits the plant enzyme nitrate reductase and there appears to be no selectivity between plant 14-3-3 isoforms in fact yeast and human isoforms appear to work equally as well in vitro. The best example where selectivity has been demonstrated is human 14-3-3o. 14-3-3o Preferential homodimerizes with itself and crystallization revealed a structural basis for this isoform s dimerization properties as well as for its specific selectivity for target binding proteins. Here partner specificity is the result of amino acid differences outside of the phosphopeptide-binding cleft. 

It has been shown [18] that when high cone-voltages were used in conjunction with negative-ion electrospray, phosphopeptides produce diagnostic ions at m/z 63 (P02 ) and m/z 79 (P03 ). LC-MS analysis of a trypsin digest of bovine... 

Casein-derived phosphorylated peptides are believed to enhance the bioavailability of calcium from milk and dairy products (Pihlanto and Korhonen, 2003), and a phosphopeptide derived from (3-casein has been shown to increase iron bioavailability (Bouhallab et ah, 2002 Peres, 1999). Other casein-derived peptides have been found to contain antihypertensive activity in rats (Leclerc et ah, 2002 Miguel et ah, 2009). A number of casein fragments demonstrate antibacterial activity (Kilara and Panyam, 2003). 

Bouhallab, S., Cinga, V., Ait-Oukhatar, N., Bureau, F., Neuville, D., Arhan, P., Maubois, J. L., and Bougie, D. (2002). Influence of various phosphopeptides of caseins on iron absorption. J. Agric. Food Chem. 50, 7127-7130. 

Li, S. and Dass, C., Iron(III)-Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry for the Purification and Characterization of Synthetic Phosphopeptides, Anal. Biochem., 270, 9, 1999. 

Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, Neel BG, Birge RB, Fajardo JE, Chou MM, Hanafusa H, Schaffhausen B, Cantley LC. SH2 domains recognize specific phosphopeptide sequences. Cell 1993 72 767-778. 

Bibbins KB, Boeuf H, Varmus HE. Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides. Mol Cell Biol 1993 13 7278-7287. 

The following diphosphates and triphosphates can be prepared by reaction of nucleotide azolides with nucleotides, sugar phosphates, phosphopeptides, or aminohexyl phosphates, a) NAD-analogues and P1, / -dinucleoside-S -diphosphates 801... 

Another means of moving beyond pure protein preparations to high-throughput characterization of proteomes is to enrich for phosphopeptides from complex mixtures by metal affinity chromatography (Andersson and Porath, 1986). Using this method, protein mixtures are proteolyzed to create peptides and phosphorylated peptides are enriched by metal affinity chromatography and subsequently identified by mass spectrometry. This method is limited, however, because in many cases phosphopeptides absorb poorly or nonphosphorylated peptides absorb nonspecifically to the metal affinity resins (Ahn and Resing, 2001). 

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

Pinkse, M.W., Uitto, P.M., Hilhorst, M.J., Ooms, B., Heck, A.J. (2004).. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal. Chem. 76, 3935-3943. 

Weng, Q.-P., Kozlowski, M., Belham, C., Zhang, A., Comb, M.J., and Avruch, J. (1998). Regulation of the p70 S6 kinase by phosphorylation in vivo Analysis using site-specific anti-phosphopeptide antibodies. J. Biol. Chem. 273, 16621-16629. 

Y. Ma, Y. Lu, H. Zeng, D. Ron, W. Mo, T. A. Neubert, Characterization of phosphopeptides from protein digests using matrix assisted laser desorption/ionization time of flight mass spectra metry and nanoelectrospray quadrupole time of flight mass spectrometry, Rapid Commun. Mass Spectrom., 15, 1693 1700 (2001). 

The specificity profile of individual SH2 domains was determined in a series of studies employing degenerate phosphopeptide library screens [53, 98] from which two consensus motifs emerged for SH2 domain binding [52-55,99,100]. One group of SH2 domains preferentially binds to a pTyr-Glu-Glu-Ile motif that defines a generic recognition sequence ... 

R. S. Annan and S. A. Carr. Phosphopeptide Analysis by Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry. Anal. Chem., 68(1996) 3413-3421. 

Chemoenzymatic Synthesis of a Characteristic Glyco-phosphopeptide from the Transactivation Domain of the Serum Response Factor, J. Sander H. Waldmann, Angew. Chem 1999, 111, 1337-1339, Angew. Chem Int. Ed. 1999,38,1250-1252... 

JS McMurray, DR Colman IV, W Wang, ML Campbell. The synthesis of phosphopeptides. Biopolymers (Pept Sci) 60, 3-31, 2001. 

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