Development of an Indole-Based Chemically Cleavable Linker Concept for Immobilizing Bait Compounds for Protein Pull-Down Experiments

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.