Department of Chemistry and the Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, NY 11794, USA.
Biochem Biophys Res Commun. 2013 Mar 8;432(2):287-90. doi: 10.1016/j.bbrc.2013.01.115. Epub 2013 Feb 8.
Protein phosphorylation is the most widely studied post-translational modification. Reversible protein phosphorylation is implicated in the regulation of a broad range of cellular processes. As such, there is extensive interest in simple and sensitive procedures for the isolation and detection of phosphorylated proteins. Synthetic analogues of ATP, with a biotin linked to the gamma-phosphate of ATP, have been reported to biotinylate kinase substrates in a kinase-catalyzed reaction. This could be an extremely attractive and versatile method for affinity enrichment of phosphorylated proteins. However, as we report here, the commercially available biotin-ATP analogue, ATP-γ-Biotin-LC-PEO-amine, is capable of biotinylating proteins independent of kinase activity. In fact, we demonstrate that this reagent is capable of non-specifically biotinylating any protein. Although the mechanism of biotinylation is not known, this report uncovers a flaw in a commercially available reagent and also highlights the importance of control experiments when developing new biochemical tools to study enzyme activity.
蛋白质磷酸化是最广泛研究的翻译后修饰。可逆蛋白质磷酸化与广泛的细胞过程的调节有关。因此，人们对简单而灵敏的方法来分离和检测磷酸化蛋白质有着浓厚的兴趣。与 ATP 相连的生物素连接到 ATP 的γ-磷酸的合成类似物已被报道在激酶催化反应中使激酶底物生物素化。这可能是一种非常有吸引力和多功能的方法，用于亲和富集磷酸化蛋白质。然而，正如我们在这里报告的那样，市售的生物素-ATP 类似物，ATP-γ-生物素-LC-PEO-胺，能够独立于激酶活性生物素化蛋白质。事实上，我们证明该试剂能够非特异性地生物素化任何蛋白质。尽管生物素化的机制尚不清楚，但本报告揭示了市售试剂的一个缺陷，并强调了在开发用于研究酶活性的新生化工具时进行对照实验的重要性。
