A targeted quantitative proteomics strategy for global kinome profiling of cancer cells and tissues

Abstract

Kinases are among the most intensively pursued enzyme superfamilies as targets for anti-cancer drugs. Large data sets on inhibitor potency and selectivity for more than 400 human kinases became available recently, offering the opportunity to design rationally novel kinase-based anti-cancer therapies. However, the expression levels and activities of kinases are highly heterogeneous among different types of cancer and even among different stages of the same cancer. The lack of effective strategy for profiling the global kinome hampers the development of kinase-targeted cancer chemotherapy. Here, we introduced a novel global kinome profiling method, based on our recently developed isotope-coded ATP-affinity probe and a targeted proteomic method using multiple-reaction monitoring (MRM), for assessing simultaneously the expression of more than 300 kinases in human cells and tissues. This MRM-based assay displayed much better sensitivity, reproducibility, and accuracy than the discovery-based shotgun proteomic method. Approximately 250 kinases could be routinely detected in the lysate of a single cell line. Additionally, the incorporation of iRT into MRM kinome library rendered our MRM kinome assay easily transferrable across different instrument platforms and laboratories. We further employed this approach for profiling kinase expression in two melanoma cell lines, which revealed substantial kinome reprogramming during cancer progression and demonstrated an excellent correlation between the anti-proliferative effects of kinase inhibitors and the expression levels of their target kinases. Therefore, this facile and accurate kinome profiling assay, together with the kinome-inhibitor interaction map, could provide invaluable knowledge to predict the effectiveness of kinase inhibitor drugs and offer the opportunity for individualized cancer chemotherapy.

Fig. 1.

Global kinome profiling with the use of isotope-coded ATP affinity probe (ICAP) and multiple-reaction monitoring (MRM). A, The structure of the ICAP probe. B, A schematic diagram showing the general workflow for MRM analysis of global kinome using ICAP.

Fig. 2.

Targeted protein kinases mapped in the dendrogram of the human kinome and linearity of iRT versus measured RT on different instruments and experimental platforms. A, Mapping of the identified protein kinases to the human kinome dedrogram. B, iRT values predict measured RT in on-line 2D experiment on Orbitrap Velos (180 min linear gradient) with an excellent correlation coefficient (R² = 0.999). C, iRT values predict measured RT in MRM experiment on TSQ Vantage (130 min linear gradient) with an excellent correlation coefficient (R² = 0.999).

Fig. 3.

MRM-based kinome assay exhibits better sensitivity, reproducibility compared with data-dependant shotgun proteomics. A, The Venn diagrams showing the overlap of quantified kinases from cell lysates of two melanoma cells obtained from MRM analysis and shotgun proteomics. B, The Venn diagrams showing the overlap of quantified kinase peptides from cell lysates of two melanoma cells by MRM analysis and shotgun proteomics. C, The Venn diagrams showing the overlap of quantified kinase peptides from cell lysates of two melanoma cells from two replicates of MRM analysis. D, The Venn diagrams showing the overlap of quantified kinase peptides from cell lysates of two melanoma cells from two replicates of shotgun proteomics experiments.

Fig. 4.

MRM-based kinome assay provided robust quantification results. A, Linear regression comparing quantification results of abundant kinase peptides from two melanoma cells obtained by MRM assay and shotgun proteomics analysis. B, Quantitative results by MRM assay for peptide ETSVLAAAK#VIDTK from SLK kinase: Extracted-ion chromatograms for three transitions monitored for light-labeled (Red) and heavy-labeled (Blue) peptides ETSVLAAAK#VIDTK in forward (Left) and reverse (Middle) labeling reactions; The consistent distribution of the peak area observed for each monitored transition from light- and heavy- labeled peptides in both forward and reverse labeling reaction along with the theoretical distribution derived from MS/MS spectra store in MRM kinome library (Right).

Fig. 5.

Quantitative comparison of kinome expression for WM-115 and WM-266–4 cells. Blue bar denotes kinase that is up-regulated in WM-266–4 cells; red bar denotes kinase that is up-regulated in WM-115 cells.

Fig. 6.

Eph tyrosine family kinases and CDKs are differentially expressed in WM-115 and WM-266-4 cells, which confers distinct sensitivities of the two lines of cells toward kinase inhibitors. A, Representative quantitative results by MRM assay in forward labeling reaction for peptide VLEDDPEATYTTSGGK#IPIR from EphA2, FLEDDTSDPTYTSALGGK#IPIR from EphB2, and FLEENSSDPTYTSSLGGK#IPIR from EphB3; peptide DLK#PNLLIDDK from CDK1, and DLK#PQNLLINTEGAIK from CDK2. Extracted ion chromatograms of light-labeled peptides from WM-115 cells are depicted in red and extracted ion chromatograms of heavy-labeled peptides from WM-266–4 cells are depicted in blue. B, Total cell lysates of WM-115 and WM-266–4 were immunoblotted with antibodies recognizing EphA2 (left) and CDK2 (right), where actin served as a loading control. C, Cell viability of WM-266–4 (blue line) and WM-115 cells (red line) when treated with dasatinib (left panel) and flavopiridol (right panel).

Fig. 7.

MRM-based global kinome profiling revealed differential expression of kinases in lung tumor and adjacent normal lung tissue. A, A heatmap showing the differential expression of kinases from tumor and adjacent normal lung tissue based on R_tumor/normal ratio in two forward and one reverse labeling reactions. Dark red and white boxes designate those kinases that are up-regulated in tumor tissue and normal tissue, respectively, as indicated by the scale bar. B, Quantitative results by MRM assay for peptide DLK#PSNLLINTTCDLK from MAPK3 kinase: (Left and Middle) Extracted ion chromatograms for three transitions monitored for light-labeled (Red) and heavy-labeled (Blue) peptides in both forward and reverse labeling reaction; (Right) the consistent distribution of the peak area observed for each monitored transition from light- and heavy- labeled peptides in both forward and reverse labeling reaction along with the theoretical distribution derived from MS/MS spectra stored in MRM kinome library.