Determining consensus substrate motifs for protein kinases using mass spectrometry: Protein Kinase A

Here we present a mass spectrometry‐based approach to determine consensus substrate motifs for purified protein kinases, illustrated using murine protein kinase A catalytic α (Prkaca). We incubate dephosphorylated proteins from multiple rat tissues with increasing concentrations of kinase under optimized conditions, followed by large‐scale phosphoproteomic profiling (trypsinization, phosphopeptide enrichment, SCX fractionation, and analysis by LC‐MS/MS [Velos‐Orbitrap]). Three search algorithms are used to assign peptide sequences to spectra. Using custom software, the sequences are aligned relative to the phosphorylated amino acid at position 0, and an information‐theoretic algorithm is used to generate position‐weighted matrices. Amino acids enriched relative to background at specific positions are displayed using sequence logos. Using our approach, we identified a Prkaca substrate preference for the sequence X‐(R/K)‐(R/K)‐X‐(pS/pT)‐Φ‐X, confirming preference for substrates with a hydrophobic amino acid (Φ) in position +1 as well as basic amino acids at −2 and −3. The method is being used to identify substrate specificities for kinases known to be involved in blood pressure control in the kidney.