Development of an indole-based chemically cleavable linker concept for immobilizing bait compounds for protein pull-down experiments |
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Authors: | Sturm Martin Leitner Alexander Lindner Wolfgang |
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Institution: | Department of Analytical Chemistry, University of Vienna, Vienna, Austria. |
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Abstract: | A novel linker chemistry based on a malondialdehyde-indole condensation reaction has been developed for the affinity-independent elution of targeted protein pull-downs. Previously developed in our lab for the tagging of tryptophan residues on proteins or peptides, the concept was extended for the design of a chemically cleavable linker system. Target molecules for interaction studies are immobilized on a solid support including the linker scaffold, and a typical pull-down experiment is carried out. After purification, the linker is cleaved by incubation with 50 mM pyrrolidine. A specific tyrosine kinase inhibitor, bosutinib, was coupled to agarose and acrylamide beads, respectively, via the new linker system, and a protein pull-down experiment of putative interaction partners from a K562 whole cell lysate was performed. The system was found to be compatible with targeted protein pull-downs; during the cleavage step, no protein hydrolysis or any degradation of amino acid side-chains was apparent. From the pull-down experiment, key targets of bosutinib such as the tyrosine kinase, Btk, were identified by liquid chromatography-tandem mass spectrometry. |
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