Table 1.
Comparison of combinatorial peptide library screening, proteome-derived library, and ProPeL motif discovery methodologies.
Figure 1.
pLogo representations of substrate sequence specificities.
pLogos for Protein Kinase A (A, B), Casein Kinase II (C, D), and control (E, F) illustrate preferred residues by position. Note, pLogos are derived from phosphorylation sites in E. coli obtained using the ProPeL methodology (after subtraction of endogenous phosphorylation sites). In each pLogo, residue heights are proportional to their log binomial probabilities in the context of the E. coli background with residues above the x-axis indicating overrepresentation and residues below the x-axis indicating underrepresentation. The central residue in each pLogo is fixed and denotes the modification site. The pLogos and corresponding extracted motifs (see Figure 2) are highly consistent with the known basophilic specificity of PKA and acidophilic specificity of CK II. Additionally, the control phosphorylation sites (i.e., endogenous E. coli phosphorylation sites) do not conform to a motif and lack any statistically significant residues.
Figure 2.
motif-x analyses for PKA (A and B) and CK II (C and D).
These motif extraction results illustrate the inter-residue correlations found among the phosphorylated peptides identified using the ProPeL methodology, and are highly consistent with the previously established consensus sequences for the PKA and CK II kinases.
Figure 3.
Goodness-of-fit of the pLogos derived from ProPeL and actual known kinase substrates versus random substrates.
Average pLogo position weight matrix scores of CK II (red) and PKA (blue) pLogos when scanned against known human substrates from the PhosphoSitePlus database compared to average scores obtained from scanning CK II and PKA pLogos against an equivalent number of random human serine and threonine residues. Error bars represent 95% confidence intervals.
Table 2.
Top 20 scan-x PKA phosphorylation predictions based on a human whole proteome scan with the PKA motif obtained using the ProPeL methodology.
Table 3.
Top 20 scan-x CK II phosphorylation predictions based on a human whole proteome scan with the CK II motif obtained using the ProPeL methodology.
Figure 4.
Receiver Operating Characteristic (ROC) curves for the scan-x and Scansite PKA and CK II kinase specific predictors.
These curves illustrate the tradeoff between sensitivity and specificity achieved by the ProPeL based scan-x (red) and combinatorial peptide library based Scansite (blue) predictors, and indicate the similarity of results achieved using these experimentally orthogonal approaches. Panels (A) and (B) are based on PKA serine and threonine predictions, respectively, while panels (C) and (D) are based on CK II serine and threonine predictions, respectively. The Scansite web server does not score all phosphorylatable residues in a given sequence, which results in partial ROC curves.