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.
Department of Analytical Chemistry, University of Vienna, Vienna, Austria.
Martin
M
Sturm
Alexander
A
Leitner
Wolfgang
W
Lindner
D000814:Aniline Compounds / Q000737:chemistry / Q000494:pharmacology; D002853:Chromatography, Liquid; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D007211:Indoles / Q000737:chemistry*; D020014:K562 Cells; D008315:Malondialdehyde / Q000737:chemistry*; D015394:Molecular Structure; D009570:Nitriles / Q000737:chemistry / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011759:Pyrrolidines / Q000737:chemistry; D011804:Quinolines / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D053719:Tandem Mass Spectrometry
D000814:Aniline Compounds; D004800:Enzymes, Immobilized; D006834:Hydrazines; D007211:Indoles; D009570:Nitriles; D047428:Protein Kinase Inhibitors; D011759:Pyrrolidines; D011804:Quinolines; C029424:hydrazine; D008315:Malondialdehyde; C471992:bosutinib; D011505:Protein-Tyrosine Kinases; C032519:pyrrolidine