Article pubs.acs.org/ac
A High-Throughput, Multiplexed Kinase Assay Using a Benchtop Orbitrap Mass Spectrometer To Investigate the Effect of Kinase Inhibitors on Kinase Signaling Pathways Ryan C. Kunz,† Fiona E. McAllister,† John Rush,‡ and Steven P. Gygi*,† †
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, United States Cell Signaling Technology, Danvers, Massachusetts 01923, United States
‡
S Supporting Information *
ABSTRACT: 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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the heavy-IStd imparts absolute quantification and site-specific localization. The inclusion of the immobilized metal affinity chromatography (IMAC) step in the original KAYAK workflow was based on the assumption that, without the enrichment of phosphopeptides, kinase activity measurements would be adversely affected because of the overwhelming abundance of substrate peptides in the reaction mix. This assumption is true for a typical phosphoproteomic experiment where the abundance of phosphopeptides remains constant after cell lysis and enzymatic digestion, and the resulting total peptide pool is dominated by nonphosphorylated peptides.16 The overwhelming number of peptides in a phosphoproteomic sample requires a fractionation step, followed by the enrichment of phosphopeptides by IMAC (or a similar enrichment strategy).16−18 In theory, the measurement of kinase activities should not be handicapped by the same technical barriers as a phosphoproteomic experiment, because of enzymatic amplification of the phosphopeptide signal. We omitted the IMAC step and found that direct analysis of phosphopeptides following the kinase reaction (“direct-KAYAK”) gave similar performance. In addition to removing the IMAC step from the assay, we reduced the number of peptide substrates from 90 to 60, while expanding the numbers of kinases/pathways covered by the
hosphorylation modulates many physiochemical properties of a protein’s activity, stability, localization, and protein− protein or protein−small-molecule interactions.1,2 Regulating the phosphorylation state of proteins involved in signaling is paramount to proper cell growth and division. Aberrant kinase signaling from gain or loss of function mutations is welldocumented as a driving force behind many diseases, most notably cancer.3−5 The entrenchment of abnormal kinase signaling in oncogenesis and other diseases makes this enzyme class a prime target for pharmaceutical intervention. Measuring kinase activities rather than simply kinase abundance is important to understand information flux though complex cellular signaling cascades.6,7 Toward this end, a simple, highthroughput, multiplexed method for measuring endogenous kinase activities is needed to better understand the relationship between kinase activities, cellular state, and phenotype. Multiple strategies exist for measuring kinase activity,6,8−13 but the mass spectrometry (MS)-based KAYAK assay uniquely combines substrate competition and site specificity. KAYAK was first described as an in vitro assay consisting of 90 “light” peptide substrates, each containing at least one possible phosphorylation site and a complementary same-sequence heavy-phosphopeptide (heavy-IStd) reference peptide set.14,15 After the kinase reaction is quenched, a known amount of heavy-IStd peptide is spiked into the quenched reaction mixture. The similar physiochemical properties of the phosphorylated light and heavy-IStd peptides allow for identical coelution, but they register in the mass spectrometer as different m/z values, because of the incorporation of a stable isotope labeled amino acid in the heavy-IStd. The presence of © 2012 American Chemical Society
Received: May 8, 2012 Accepted: June 19, 2012 Published: June 19, 2012 6233
dx.doi.org/10.1021/ac301116z | Anal. Chem. 2012, 84, 6233−6239
Analytical Chemistry
Article
Figure 1. Workflow for direct-KAYAK and KAYAK. 60 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 asterisks. 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 the letters A, B, and C.
columns were washed with 200 μL 0.1% TFA (twice). Desalted peptides were elute into a clean 96-well microtiter plate, using 50 μL of 50% ACN + 0.1% FA. The eluted peptides were dried down in a speedvac evaporator or covered and left in the 37 °C warm room overnight. Once dried, the peptides from directKAYAK samples were resuspended in 100 μL of 5% formic acid and directly analyzed using liquid chromatography−mass spectrometry (LC-MS). Peptides from KAYAK samples were further processed, using IMAC resin for phosphopeptide enrichment. IMAC Step. Desalted peptides from KAYAK assays were mixed with 10 μL of IMAC resin pre-equilibrated with 40% ACN + 25 mM FA. The peptide/IMAC slurry is incubated at 25 °C for 1 h; then transferred to a StageTip packed with four “cookies” of Empore disk C18 material.20 The stacked IMACC18 StageTip was washed twice with 50 μL of 40% ACN + 25 mM FA, followed by 40 μL of 0.1% TFA. Bound phosphopeptides were eluted from the IMAC resin to the C18 cookies with two 100-μL washes of 500 mM K2HPO4, pH 7.0. The C18 cookies containing the phosphopeptides were washed with 20 μL of 0.1% TFA, followed by 20 μL of 1% FA. Phosphopeptides are eluted from the C18 cookies using 20 μL of 50% ACN + 1% AA; this step is performed twice. The enriched phosphopetides were dried in a speedvac evaporator. Once dry, the phosphopeptides were resuspended in 10 μL of 5% FA and analyzed using LC-MS. It is worth noting that the peptides from the KAYAK workflow were resuspended in 10 μL of 5% FA, whereas the peptides from the direct-KAYAK workflow were resuspended in 100 μL of 5% FA (see previous section). We have measured a 10% recovery, following the IMAC step (data not shown), and therefore have adjusted the final sample volume for LC-MS analysis accordingly. The volume adjustment of the final sample before introduction into the LC-MS takes into account the loss of peptides from the IMAC step. In both the KAYAK and direct-KAYAK samples, a volume of 2 μL was injected for LC-MS analysis. Western Blotting. Lysates (15−20 μg of total protein) were separated by SDS-PAGE (NuPAGE 4−12% Bis-Tris 1.5 mm, 15 Well). Proteins were transferred to a nitrocellulose membrane using the iBlot system. Blots were blocked for 1 h at room temperature with 5% nonfat dry milk in TBST. Following blocking, blots were probed with primary antibody overnight at
assay. The direct-KAYAK assay is now appropriate for a 96-well plate platform. Using kinase inhibitors, we measured dosedependent inhibition of important kinase signaling pathways in the monocytic leukemia cell line THP-1.19 Using the inhibitor data, we calculated IC50 values from the kinase targeted by the inhibitor and also kinases downstream of the intended target.
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EXPERIMENTAL SECTION The Equipment, Chemicals and Reagents, and Cell Culture and Lysis sections can be found in the Supporting Information section. Direct-KAYAK and KAYAK Assay. Detailed descriptions of the KAYAK assay and workflow, along with peptide synthesis, purification, and quantification, were described earlier.14,15 In brief, direct-KAYAK and KAYAK assays are performed using 10 μg of total protein, 2 μM of each peptide substrate (60 different substrate peptides were used, see Table S1 in the Supporting Information for a list of sequence and selected details), 1× KAYAK reaction buffer (10× KAYAK reaction buffer consist of: 250 mM TRIS-HCl pH 7.5, 50 mM ATP, 75 mM MgCl2, 2 mM EGTA, 75 mM β-glycerophosphate, 1 mM sodium vanadate, and 1 mM DTT). KAYAK reactions were performed in a total volume of 25 μL at 25 °C for 30 min. Reactions were quenched with 50 μL 1% TFA, and 5 pmol of each heavy-IStd was added, following the quench step. It is important to note that the 10× KAYAK reaction buffer used for the inhibition studies contained 500 μM ATP. The concentrations of other components in the reaction buffer remain the same as outlined above. This point is important because the kinase inhibitors used in this study are ATPcompetitive. If the ATP concentration in the assay is too high, the apparent IC50 values will appear higher than their true values, because of competition between ATP and the inhibitor. Desalting and Peptide Workup. Both direct-KAYAK and KAYAK samples were desalted using a 10 mg SPE C18 μElution 96-well plate platform. The C18 μElution columns were washed with 200 μL of 100% MeOH, followed by 200 μL of 50% ACN + 0.1% TFA and an equilibration step of 200 μL of 0.1% TFA. Once equilibrated, the quenched KAYAK reactions were applied to the C18 μElution columns. The C18 μElution 6234
dx.doi.org/10.1021/ac301116z | Anal. Chem. 2012, 84, 6233−6239
Analytical Chemistry
Article
4 °C. Primary antibodies were diluted 1/2000 in TBST + 5% BSA. Blots were probed for 1 h at room temperature with secondary HRP conjugated goat antirabbit diluted 1/20 000 in TBST + 2% BSA. Blots were visualized using Femto ECL chemiluminescence as recommended by the manufacturer. Liquid Chromatography and Mass Spectrometry. KAYAK and direct-KAYAK analysis was performed on an Exactive-Orbitrap mass spectrometer equipped with a Thermo Fisher nanospray source, a PAL HTC autosampler for sample handling, and an Accela HPLC pump for liquid chromatography (LC) separations. Nanoliter flow rates across the column were achieved using a flow-split method. Peptides were separated on a 125 μm × 18 cm hand-pulled fused silica microcapillary column with a needle tip diameter of
