Kinase-substrate recognition depends on the chemical properties of the phosphorylatable residue as well as the surrounding linear sequence motif. Detailed knowledge of these characteristics increases the confidence of linking identified phosphorylation sites to kinases, predicting phosphorylation sites, and designing optimal peptide substrates. Here, we present a mass spectrometry-based approach for determining linear kinase substrate motifs by elaborating the positional and chemical preference of the kinase for a phosphorylatable residue using libraries of naturally-occurring peptides that are amenable to peptide identification by commonly used proteomics platforms. We applied this approach to a structurally and functionally diverse set of purified kinases, which recapitulated their previously described substrate motifs and discovered additional ones, including preferences of certain kinases for phosphorylatable residues adjacent to peptide termini. Furthermore, we identify specific and distinguishable motif elements for the four members of the polo-like kinase (Plk) family and verify members of these motif elements for Plk1 in vivo.
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