2014
DOI: 10.1039/c3md00315a
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Rapid profiling of protein kinase inhibitors by quantitative proteomics

Abstract: The ability to determine structure-activity relationships (SAR) and identify cellular targets from cell lysates and tissues is of great utility for kinase inhibitor drug discovery. We describe a streamlined mass spectrometry-based chemoproteomics workflow to examine the SAR and target profiles of a small library of kinase inhibitors that consists of the drug dasatinib and a panel of general type II inhibitors. By combining a simplified affinity enrichment and on-bead protein digestion workflow with quantitativ… Show more

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Cited by 21 publications
(29 citation statements)
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“…1a). 5, 24, 28, 3335 The inhibitor analogs 1 and 3–5 were described previously and the affinity reagents 2 , 6 and 7 were newly developed in our laboratory. Compounds 1–7 were immobilized on carboxy-derivatized sepharose using amide coupling chemistry to yield the corresponding affinity matrices.…”
Section: Resultsmentioning
confidence: 99%
See 1 more Smart Citation
“…1a). 5, 24, 28, 3335 The inhibitor analogs 1 and 3–5 were described previously and the affinity reagents 2 , 6 and 7 were newly developed in our laboratory. Compounds 1–7 were immobilized on carboxy-derivatized sepharose using amide coupling chemistry to yield the corresponding affinity matrices.…”
Section: Resultsmentioning
confidence: 99%
“…1b). 5, 18 Experiments were done in quadruplicate, with two SILAC label swap experiments in which SILAC labels were switched between control and inhibitor competition experiments. The label swap experiment helps to distinguish true hits from non-selective hits as the former show an inversion of the sign of the log2 SILAC ratios, while contaminants and false positives do not.…”
Section: Resultsmentioning
confidence: 99%
“…This number is in agreement with previously reported dasatinib enrichment experiments. 15 Of the four additional proteins with a Heavy/Light ratio > +1 σ, three (HNRPF, ARPC3, and RL37A) were very close to the cut-off. The remaining protein, NOMO3, is a poorly annotated single-pass membrane protein that has not previously been reported to interact with dasatinib.…”
mentioning
confidence: 89%
“…The seven kinobead affinity reagents used were synthesized in-house as described previously (56,57). For optimal coverage of the human kinome an optimized mixture of the seven kinobead reagents was prepared as follows: 1 ml of reagent 1, 0.5 ml of reagents 2, 3 and 7, respectively, and 0.25 ml of reagents 4, 5 and 6, respectively, to give 3.25 ml of the complete kinobead mixture.…”
Section: Preparation Of Optimized Kinobead Mixturementioning
confidence: 99%