Phosphopeptides synthetic, analysis

Competition binding analysis of a variety of phosphopeptides derived from different Cdc4 substrates revealed a range of affinities. For example. Sic IP = 24 pM, FarlP = 2.7 pM, Gcn4 = 0.8 pM. Importantly, multi-site phosphorylation dependent interactions with Cdc4 can be recapitulated with synthetic concatamers of low affinity CPD sites based on either the regions T45 or S76 sites in Sicl. 

The last four studies performed by the PSRG have been selected as examples of comparative peptide analysis for this section on the analytics of synthetic peptides. These include techniques for the synthesis and production of cyclic peptides and phosphopeptides and include studies on the potential problems of racemization and deamidation 10 1516191 These studies were selected for the following reasons first, the cyclized and phosphorylated forms of peptides are synthesized and used by many scientists secondly, these studies also address potential problems that can arise using these techniques and thirdly, these studies are provided as references in which readers might find useful methods for each type of analysis. 

The use of isotope-labelled synthetic peptides as IS was proposed for the absolute quantification of proteins in protein expression studies [113]. If necessary, these synthetic peptides can be covalently modified to be applied as IS in for instance phosphopeptide quantification. This approach was applied in the quantitative analysis of two glutathione 5 -transferase isoforms in human liver cytosol by LC-MS in SRM mode [114]. A series of pilot experiments were performed to select the most suitable IS peptides for four human plasma proteins (hemopexin, ocl antichymotrypsin, interleiddn-6, and tumor necrosis factor-oc) [115]. Rabbit polyclonal antibodies were raised against these selected peptides and immobilized on POROS supports. These lAC columns were applied to achieve a 120-fold emichment of the antigen peptide from digested human plasma proteins. The peptides and its IS were measured by LC-MS in SIM or SRM mode. The methods appears to be a tailor method for targeted protein analysis. 

Fig. 3 Synthetic phosphopeptides representing sequences around Ros Y2267 and pY2327 were analyzed for binding of the JV-terminal SH2 domain of SHP-1 by surface plasmon resonance. Representative experiments are shown (left panel). The data were fitted to determine the kinetic constants ka and k. Signals at equilibrium were also fit using an equilibrium analysis to determine the affinities of the interactions (right panels). Reproduced from [10] with permission from the Company of Biologists 2004...

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