Crizotinib Affinity Probe Preparation

The crizotinib affinity probe was prepared in two steps from commercially available 3-[(1RS)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyridin-2-amine (Selleckchem) according to literature and drug-affinity matrices were prepared as described previously (Huber et al., 2014). Briefly, affinity chromatography and elution were performed in duplicate using 25nmol of compound immobilized on 50μL NHS-activated Sepharose 4 Fast Flow beads (GE Healthcare Bio-Sciences) and 10mg total cell lysate as protein input per replicate. For competition experiments, cell lysates were pretreated with 20μM 3-[(1RS)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyridin-2-amine for 30 minutes. Eluates were labeled with iTRAQ and quantitative protein mass spectrometry and bioinformatics analysis utilizing the R isobar package were performed as previously reported 48 (link),52 (link).

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Vladimer G.I., Snijder B., Krall N., Bigenzahn J.W., Huber K.V., Lardeau C.H., Sanjiv K., Ringler A., Berglund U.W., Sabler M., de la Fuente O.L., Knöbl P., Kubicek S., Helleday T., Jäger U, & Superti-Furga G. (2017). Global survey of the immunomodulatory potential of common drugs. Nature chemical biology, 13(6), 681-690.

Publication 2017

Sepharose 4 Fast Flow beads

1h pyrazol Affinity chromatography Amine Cell Crizotinib Drug Mass spectrometry Protein Replicate Sepharose

Corresponding Organization : Medical University of Vienna

Other organizations : CeMM Research Center for Molecular Medicine, Austrian Academy of Sciences, Science for Life Laboratory, Karolinska Institutet

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Variable analysis

independent variables

Concentration of 3-[(1RS)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyridin-2-amine used for competition experiments (20 μM)

dependent variables

Proteins bound to the crizotinib affinity probe

control variables

Amount of compound immobilized on NHS-activated Sepharose 4 Fast Flow beads (25 nmol)
Amount of total cell lysate used as protein input per replicate (10 mg)
Experimental setup (affinity chromatography and elution performed in duplicate)

positive controls

None specified

negative controls

Competition experiments with 20 μM 3-[(1RS)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyridin-2-amine

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