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Oct 29, 2012

A Faster KAYAK: Mass Spectroscopy for Multiplexed Kinase Assays

Researchers developed a new, simplified method for measuring the phosphorylation rates of peptide substrates.

A Faster KAYAK: Mass Spectroscopy for Multiplexed Kinase Assays

The direct-KAYAK method can be run in a relatively high-throughput 96-plate format with a benchtop orbitrap mass spectrometer. [Stefan Rajewski - Fotolia.com]

  • Click Image To Enlarge +
    Figure 1. Workflow for direct-KAYAK and KAYAK. Sixty light synthetic substrate peptides are incubated with a sample containing active kinase(s). The kinase reaction is quenched with acid and heavy stable isotope-labeled phosphopeptide internal standards (heavy-Istd) are added; heavy is denoted by an asterisk. The reaction is then desalted on a 96-well C18 SPE plate. Following the desalting step the peptides are either directly analyzed by LC-MS (direct-KAYAK) or enriched for phosphopeptides using IMAC resin (KAYAK, red arrows). MS data are collected on an Exactive-orbitrap mass spectrometer and analyzed using Pinpoint software. The three major steps where improvements were made to the workflow are indicated by A, B, and C. KAYAK, kinase activity assay for kinome profiling; LC-MS, liquid chromatography–mass spectroscopy; IMAC, immobilized metal affinity chromatography.

    Kinases have been the target of extensive research, and there has been significant interest in developing assays that measure the endogenous activities of kinases and their perturbation by small molecules. Many small molecule kinase inhibitors have extensive polypharmacology, so it is important to understand the pathways and off-targets that a potential new kinase inhibitor affects in vivo.

    Kinase activity assay for kinome profiling (KAYAK) is a method that allows the phosphorylation of a diverse set of kinase peptide substrates to be simultaneously prosecuted. This new article* describes a modification to the technique, called direct-KAYAK, wherein the substrate set (light substrate peptides) was modified, the phosphopeptide enrichment step was removed, and the data analysis was streamlined (see Figure 1). The method can be run in a relatively high-throughput 96-plate format with a benchtop orbitrap mass spectrometer.

    The heavy-phosphopeptides that are added following the acid quench step are complementary in sequence to the light peptides and have similar physicochemical properties, allowing them to co-elute with the light peptides but to be distinguished by their different m/z ratios in the mass spectrometer. This enables quantification and site-specific localization.

  • Click Image To Enlarge +
    Figure 2. Dose-dependent inhibition values can be measured from an unfractionated cell lysate using direct-KAYAK. Data from four biological replicates of THP-1 cells treated with Ro-31-8425, followed by PMA stimulation were fit with a sigmoidal dose-dependent equation for peptides (A) B2 and (B) E11. Error bars represent SEM (n = 4) values. The observed IC50 value is shown in the inset. (C) Western blot analysis of kinases involved in the phosphorylation of peptide E11 (RSK activity) from assays using THP-1 cells treated with Ro-31-8425. Antibodies used are indicated left of the blots. Treatment information is indicated above the blot.

    The authors showed that while the direct-KAYAK does have unreacted substrate peptides in contrast to the KAYAK method that contains the phosphopeptide enrichment step, the phosphopeptide signal can still be accurately measured. Kinases that phosphorylate the set of substrates used in the method include protein kinase A, B, and C; mitogen-activated kinases; cyclin-dependent kinases; AMP-activated protein kinase; DNA-damage checkpoint kinase; glycogen synthase kinase; and ABL1 (Abelson murine leukemia viral oncogene homolog 1) kinase and FAK (focal adhesion kinase) kinase. The response to inhibitors was tested in THP-1 cells, a model system whereby monocyte to macrophage differentiation can be studied by treating the cells with the protein kinase C activator PMA (phorbol 12-myristate 13-acetate). Ro-31-8425, a potent and selective inhibitor of protein kinase C, showed inhibition of phosphorylation for many of the substrate peptides, including substrate peptides for RSK (ribosomal s6 kinase) and protein kinase C (see Figure 2).

    KAYAK and direct-KAYAK are an interesting addition to the field of proteomics. Because there are over 400 protein kinases in the human kinome, it is important to realize the limitation of using a small subset of kinase substrates to extrapolate to overall kinase selectivity, and the use of larger commercially available selectivity screens to assess kinase selectivity would still be recommended.

  • *Abstract from Analytical Chemistry 2012, Vol. 84: 6233–6239

    Protein phosphorylation is an important and ubiquitous post-translational modification in eukaryotic biological systems. The KAYAK (kinase activity assay for kinome profiling) assay measures the phosphorylation rates of dozens of peptide substrates simultaneously, directly from cell lysates. Here, we simplified the assay by removing the phosphopeptide enrichment step, increasing throughput while maintaining similar data quality. We term this new method direct-KAYAK, because kinase activities were measured directly from reaction mixtures after desalting. In addition, new peptides were included to profile additional kinase pathways and redundant substrate peptides were removed. Finally, the method is now performed in 96-well plate format using a benchtop orbitrap mass spectrometer and the Pinpoint software package for improved data analysis. We applied the new high-throughput method to measure IC50 values for kinases involved in monocyte-to-macrophage differentiation, a process important for inflammation and the immune response.


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