Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes.
Qian ZhaoXiaohu OuyangXiaobo WanKetan S GajiwalaJohn C KathLyn H JonesAlma L BurlingameJack TauntonPublished in: Journal of the American Chemical Society (2017)
Protein kinases comprise a large family of structurally related enzymes. A major goal in kinase-inhibitor development is to selectively engage the desired kinase while avoiding myriad off-target kinases. However, quantifying inhibitor interactions with multiple endogenous kinases in live cells remains an unmet challenge. Here, we report the design of sulfonyl fluoride probes that covalently label a broad swath of the intracellular kinome with high efficiency. Protein crystallography and mass spectrometry confirmed a chemoselective reaction between the sulfonyl fluoride and a conserved lysine in the ATP binding site. Optimized probe 2 (XO44) covalently modified up to 133 endogenous kinases, efficiently competing with high intracellular concentrations of ATP. We employed probe 2 and label-free mass spectrometry to quantify intracellular kinase engagement by the approved drug, dasatinib. The data revealed saturable dasatinib binding to a small subset of kinase targets at clinically relevant concentrations, highlighting the utility of lysine-targeted sulfonyl fluoride probes in demanding chemoproteomic applications.
Keyphrases
- living cells
- mass spectrometry
- drinking water
- induced apoptosis
- small molecule
- protein kinase
- high efficiency
- label free
- amino acid
- cell cycle arrest
- tyrosine kinase
- liquid chromatography
- single molecule
- reactive oxygen species
- fluorescence imaging
- fluorescent probe
- protein protein
- single cell
- quantum dots
- cancer therapy
- endoplasmic reticulum stress
- capillary electrophoresis
- electronic health record
- social media
- high performance liquid chromatography
- nucleic acid
- oxidative stress
- gas chromatography
- binding protein
- big data
- emergency department
- cell proliferation
- chronic myeloid leukemia
- machine learning
- data analysis
- deep learning