Phosphopeptide Isolation

Exterrsion of the above procedure trsing ethanedithiol, enabling subsequent biotirtylation to obtain a phosphoprotein isotope-coded affinity tag (PhlAT) [53-54]. This errables phosphopeptide isolation by avidin affinity chromatography. 

Nuwaysii, E.M. Stults, J.T. "Electrospray Ionization Mass Spectrometry of Phosphopeptides Isolated by On-Line Immobilized Metal-Ion Affinity Chromatography," J. Am. Soc. Mass Spectrom. 4, 662-669 (1993). 

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. 

Ellegard, K.H., Gammelgard-Larsen, C., Sorensen, E.S., and Fedosov, S. 1999. Process scale chromatographic isolation, characterization and identification of tryptic bioactive casein phosphopeptides. Int. Dairy J. 9, 639-652. 

Gagnaire, V., Pierre, A., Molle, D., and Leonil, J. 1996. Phosphopeptides interacting with colloidal calcium phosphate isolated by tryptic hydrolysis of bovine casein micelles. J. Dairy Res. 63, 405-422. 

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). 

Niihse T, Yu K, Salomon A. Isolation of phosphopeptides by immobilized metal ion affinity chromatography. Curr. Protoc. Mol. Biol. 2007 18 18.13. 

Having isolated the phosphopeptide-containing fractions, it becomes possible to try to locate the site of phosphorylation by tandem mass spectrometry. 

Selective isolation of phosphopeptides from tiyptic digests using a dualprecolumn setup (titanium oxide and RPLC) prior to RPLC-MS was reported by Pinkse et al. [44]. The tryptic digest is injected onto two precolumns in series phosphopeptides are selectively trapped by the TiOj-column, whereas non-... 

M.B. Goshe, T.P. Comads, E.A. Panisko, N.H. Angell, T.D. Veenstra, R.D. Smith, Phosphoprotein ICAT approach for isolating and quantifying phosphopeptides in proteome-wide analyses. Anal. Chem., 73 (2001)2578. 

A general protocol for mass spectrometric analysis of phosphoproteins is illustrated in Figure 15 various steps of this protocol are cleavage of purified phosphoproteins, isolation and preferential enrichment of phosphopeptides, selective detection of phosphopeptides in the digest, identification of the phosphorylation sites using tandem mass spectrometry, and identification of phosphopeptides/proteins through a database search. 

Another revolutionary idea in the enrichment of phosphopeptides is to attach unique chemical tags to the peptides.95 96 In one approach, applicable to phosphoserine- and phosphothreonine-containing peptides, the phosphate group is replaced with 1,2-ethanedithiol, and biotin is attached to the thiol group for selective isolation of phosphopeptides by avidin column chromatography.95 A chemical tag that is applicable to all three phospho residues has been developed 96 The enrichment of phosphopeptides in peptide digests has also been achieved with calcium phosphate precipitation.97... 

Goshe, M.B. Conrads, T.P. Panisko, E.A. Angell, N.H. Veenstra, T.D. Smith, R.D. Phosphoprotein Isotope-Coded Affinity Tag Approach for Isolating and Quantifying Phosphopeptides in Proteome-wide Analyses, Anal. Chem. 73, 2578-2586 (2001). 

Two-dimensional phosphopeptide maps of LC20 from 32p-iabeled muscles stimulated with different agents allows one to differentiate between the involvement of MLCK and PKC the phosphopeptide map of LC20 isolated from K+-stimulated arteries shows predominantly MLCK/Ser peptides (Erdodi et al, 1987), whereas in phorboldibutyrate (PDBu)-treated muscle... 

FIGURES Autoradiograms of phosphopeptide maps of isolated LC20 phosphorylated by either MLCK or PKC. From Erdodi et al. (1988a, Fig. 4, p. 587). 

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