Department of Analytical Chemistry, University of Vienna, Vienna, Austria.
Bioconjug Chem. 2011 Feb 16;22(2):211-7. doi: 10.1021/bc100330a. Epub 2011 Jan 19.
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.
一种基于丙二醛-吲哚缩合反应的新型连接化学物质已被开发出来，用于非亲和性洗脱靶向蛋白下拉实验。该化学物质先前在我们实验室中被开发用于蛋白质或肽上色氨酸残基的标记，其概念被扩展用于设计化学可切割的连接系统。用于相互作用研究的靶分子被固定在包括连接支架的固体支持物上，并进行典型的下拉实验。纯化后，通过在 50mM 吡咯烷中孵育将连接物切割。通过新的连接系统，将一种特定的酪氨酸激酶抑制剂博舒替尼分别偶联到琼脂糖和丙烯酰胺珠上，并从 K562 全细胞裂解物中进行潜在相互作用伙伴的蛋白下拉实验。该系统与靶向蛋白下拉实验兼容；在切割步骤中，没有明显的蛋白质水解或氨基酸侧链的任何降解。通过下拉实验，通过液相色谱-串联质谱鉴定了博舒替尼的关键靶标，如酪氨酸激酶 Btk。
