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Assessment of rational peptide design for kinase activity assays by mass spectrometry Marcel Stoop, Jetse Scholma, Maikel Peppelenbosch, Jos Joore and Theo Luider Introduction (max 120 words) Protein kinases are key regulators of cellular processes, including proliferation, differentiation and apoptosis. Kinase activity can be assessed by measuring the phosphorylation of peptides derived from physiological downstream substrates, for example by mass spectrometry. However, these peptide substrates are not equally suitable for use in kinase activity assays, because they lack sensitivity and/or specificity towards individual kinases. We therefore set out to develop a strategy to select peptide substrates with optimized sensitivity and specificity towards selected kinases, in order to improve peptide-based kinase assays. Increased specificity is especially important for assaying individual kinase activities in complex mixtures, such as cell lysates, containing many different protein kinases. Methods (max 120 words) As a first step towards a method for designing better peptide substrates, we used Predikin (http://predikin.biosci.uq.edu.au/) as a tool to quantify the interaction of each amino acid in a heptapeptide motif of a substrate with specific amino acids in the enzymatic cleft of a kinase, resulting in weight matrices containing scores for each amino acid in each heptapeptide position. Here, we used the combination of sensitivity matrices of two kinases merged into a specificity matrix to rationally design peptides for a kinase activity assay that could distinguish between the different, but related kinases CDK1 and MAPK1. The designed heptapeptides were synthesized as substrate peptides with the following structure: Biotin-AhxGGGG-heptapeptide-GGGG-OH. Phosphorylation reactions were carried out and measured in triplicate. Preliminary results (max 300 words) Mitogen activated protein kinase 1 (MAPK1) and Cyclin dependent kinase 1 (CDK1) were selected from the CMGC group of kinases, a group of evolutionary related proline-directed kinases. These kinases are expected to be dissimilar enough to be distinguishable in a kinase activity assay, while being similar enough to display overlapping phosphorylation patterns. Peptides (12 peptides for each kinase) were homogeneously incubated with each of the kinases separately. Phosphorylation causes a 80 Da shift in the mass of the peptide fragment, which allowed a quantification of the fraction of phosphorylated peptide and the unphosphorylated substrate peptide using mass spectrometry. Known kinase substrates display variation of phosphorylation affinities. The peptides that had been included as control peptides (natural substrate peptide seqences) based on good scores for sensitivity and specificity performed well, especially for CDK1. CDK1 control peptides that had low scores for specificity showed no phosphorylation. MAPK1 substrates with the lowest scores for specificity had notably higher sensitivity scores for CDK1 than for MAPK1 and, accordingly, are only phosphorylated by CDK1. Designed peptides without proline at position +1 showed no phosphorylation, whereas the same set with proline at position +1 performed better. The phosphorylated fractions were plotted against the corresponding Predikin score for all peptides, showing, a threshold score of 81-83 marking the division between unphosphorylated peptides (lower scores) and phosphorylated peptides (higher scores), although some peptides with higher Predikin score were not phosphorylated. Among substrates with Predikin scores >80 there is a 0.6 correlation between Predikin score and phosphorylated fraction for CDK1 and a 0.8 correlation for MAPK1. These results show that mass spectrometry can be employed to assess peptide suitability for kinase activity assays. Extending this research to more kinases would allow for the generation of Predikin score cut-off values to facilitate rational peptide design for kinase activity assays. Novel aspect The use of mass spectrometry for assessment of suitability of substrate peptides for kinase activity assays.