title: Unexpected biotinylation using ATP-γ-Biotin-LC-PEO-amine as a kinase substrate.
abstract: 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.
authors: Department of Chemistry and the Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, NY 11794, USA.
authors: Dhruv P
authors: DP
authors: Arora
authors: Elizabeth M
authors: EM
authors: Boon
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry*; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D019921:Biotinylation; D000071677:Histidine Kinase; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: C583164:ATP-gamma-Biotin-LC-PEO-amine; D011506:Proteins; D001710:Biotin; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D000071677:Histidine Kinase