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. 2009 Jun 29;106(28):11606–11611. doi: 10.1073/pnas.0905165106

Fig. 1.

Fig. 1.

General scheme of the KAYAK strategy. (A) Overview of the procedure, where 90 synthetic peptides are used as substrates for in vitro kinase assays. (B) Example of high-resolution MS and elution chromatogram for a light and heavy (internal standard; IStd) pair of phosphopeptides. Asterisk indicates incorporation of a proline residue containing 6 additional Daltons of heavy isotopes. (C) Intensity map representation of substrate phosphorylation activities (average of triplicate experiments) from 6 μg starved HEK293 cell lysates toward each of the 90 peptides in their respective plate positions. ND, not detected, indicates the ones below the threshold. (D) Immunoblot analysis of the lysates of insulin- and EGF-stimulated HEK293 cells using the indicated antibodies. (E) Examples of phosphorylation rates (performed from 3 separate experiments) for a preferred Akt (A3; RPRAAtFPFR) and RSK (B6; PKRKVsSAEGPFR) substrate, respectively, using the lysates in (D). (F) Time course of the Akt-specific substrate, A3, phosphorylation reaction using the same lysates in D.