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 anticancer 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. Molecular

Application of Adenosine Triphosphate Affinity Probe and Scheduled Multiple-Reaction Monitoring Analysis for Profiling Global Kinome in Human Cells in Response to Arsenite Treatment

“…13 The acquired tandem mass spectra and retention time information on kinase-derived peptides with desthiobiotin labeling were then processed using Skyline for the MRM library construction. However, the use of isotope-coded ATP affinity probes in the previous study required customized synthesis, which may limit its general application in analytical laboratories.…”

“…13 However, this targeted MRM analysis was not applied to study the global kinome response upon external chemical stimulus, which has broad applications in research in cell signaling and environmental toxicology. Therefore, in the current study, we further expanded the application of this technique to elucidate, with the use of an SILAC-based approach, the perturbation of the entire kinome induced by an environmental toxicant, sodium arsenite.…”

Target Identification in Small Cell Lung Cancer via Integrated Phenotypic Screening and Activity-Based Protein Profiling

“…The desthiobiotin-ATP probe covalently labels conserved lysine residues in or near the ATP binding pocket of kinases, which are then enriched, identified, and quantified by avidin-based purification of labeled peptides and LC-MS/MS. This approach is uniquely capable of profiling the human kinome in human disease or cell models and can identify biological targets of kinase inhibitors through competitive binding of the active sites of the kinase with an ATP probe (4–6). …”

Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides

“…ABPP-LC-MRM approaches with isotope-coded probes and extensive development of stable isotope-labeled standard (SIS) peptides have recently been reported. [29,30] As an example of an endogenous post-translational modification, phosphotyrosine-containing (pY) peptides can be enriched by immunoprecipitation, providing the ability to examine signaling networks and the consequences of kinase inhibitor treatment. [21,31–32] The complement of these two techniques provides additional insight and higher confidence in altered signaling pathways by combining kinase activity and substrate phosphorylation into pathway maps generated from the differentially modified peptides and their proteins of origin.…”