A full understanding of the proteome will require ligands to all of the proteins encoded by genomes. While antibodies represent the principle affinity reagents used to bind proteins, their limitations have created a need for new ligands to large numbers of proteins. Here we propose a general concept to obtain protein affinity reagents that avoids animal immunization and iterative selection steps. Central to this process is the idea that small peptide libraries contain sequences that will bind to independent regions on a protein surface and that these ligands can be combined on synthetic scaffolds to create high affinity bivalent reagents. To demonstrate the feasibility of this approach, an array of 4000 unique 12-mer peptides was screened to identify sequences that bind to nonoverlapping sites on the yeast regulatory protein Gal80. Individual peptide ligands were screened at different distances using a novel DNA linking strategy to identify the optimal peptide pair and peptide pair separation distance required to transform two weaker ligands into a single high affinity protein capture reagent. A synthetic antibody or synbody was created with 5 nM affinity to Gal80 that functions in conventional ELISA and pull-down assays. We validated our synthetic antibody approach by creating a second synbody to human transferrin. In both cases, we observed an increase in binding affinity of  ~1000-fold (ΔΔG = ~4.1 kcal/mol) between the individual peptides and final bivalent synbody construct.

Peptide Microarray Publication – Creating protein affinity reagents by combining peptide ligands on synthetic DNA scaffolds is a post from: LC Sciences Blog. More information about LC Sciences can be here.

• PKS – peptide microarray
• 27 known protein tyrosine kinase substrates and their sequence variants
• All peptides on the chip contain 8-10 redundancies.
• Positive controls – synthetic phosphopeptides (pY phosphotyrosine incorporated by synthesis)
• Negative controls – Ala substitution of Tyr at the phosphorylation site
• Src kinase, p60c-src (Invitrogen)

Assay

• Sample – Src kinase, p60c-src (Invitrogen)
• Enzyme concentration – 0.5 mg/ml
• Volume – 50 µl
• Reaction time – 30 minutes or longer

Detection

• Fluorescent dye is used to specifically stain phosphate groups (pS, pT, and pY).
• Only the phosphate group of the phosphopeptides will be specifically stained by the fluorescent dye.  No need for antibodies which are known to suffer from non-specific or weak binding problems. If specially request, antibody detection can be applied.

Results

• Figure 1 – Fluorescent image (inverted) of 4K kinase profiling microarray
• (A) Image of a subset of four replicates of the I(Y/A)GEF, pY and Y sequences – The result shows that IYGEF is phosphorylated.
• (B) Image of the YVPM (column 1) and the YEEIP (column 2) related sequences in double replicates – Among these, YEE and YEEI are phosphorylated by the Src kinase used, and YEEIP is a substrate of lower reactivitiy, and YVPM has the lowest reactivity.  Their results are consistent with the literature information.

Figure 1

Figure 2 – Plot of the relative phosphorylation efficiency of each Y-containing peptide

• The phosphorylating efficiencies of YEEI and IYGEF surpassed 90% of the synthetic pYEEI or pYGEF.
• Except YLEL, Src kinase selected peptides with Glu, Asp or amino acids with small side chains (Gly or Ala) at the P+1 position. (ratio>30%, a line placed)
• This was also demonstrated by Songyang’s paper (Songyang, Z and Cantley, L.C., (1995) Recognition and specificity in protein tyrosine kinase-mediated signaling, Trends in Biology Sciences, 20, 470-475).  [abstract]

• Relative Phosphorylation Efficiency:  fp% = (I_Y-I_A) / (I_pY-I_A)*100
• I_Y – signal intensity of phosphorylated Y-peptide
• I_pY – intensity of synthetic phosphopeptide
• I_A – intensity of Y>A substitution peptide as negative control

Application Note – Large Scale Mapping of Kinase Substrate Specificity is a post from: LC Sciences Blog. More information about LC Sciences can be here.

Peptide Microarrays Discussed in Recent GEN Article is a post from: LC Sciences Blog. More information about LC Sciences can be here.

Phosphoprotein Binding – Peptide Microarray is a post from: LC Sciences Blog. More information about LC Sciences can be here.

Minireview – Synthesis and Application of Peptide Arrays: Quo Vadis SPOT Technology is a post from: LC Sciences Blog. More information about LC Sciences can be here.