Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, USA.
Chembiochem. 2013 Feb 11;14(3):381-7. doi: 10.1002/cbic.201200626. Epub 2013 Jan 17.
Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphorylation is critical to understand cell biology. Our lab recently discovered kinase-catalyzed biotinylation, where ATP-biotin is utilized by kinases to label phosphopeptides or phosphoproteins with a biotin tag. To exploit kinase-catalyzed biotinylation for phosphoprotein purification and identification in a cellular context, the susceptibility of the biotin tag to phosphatases was characterized. We found that the phosphorylbiotin group on peptide and protein substrates was relatively insensitive to protein phosphatases. To understand how phosphatase stability would impact phosphoproteomics research applications, kinase-catalyzed biotinylation of cell lysates was performed in the presence of kinase or phosphatase inhibitors. We found that biotinylation with ATP-biotin was sensitive to inhibitors, although with variable effects compared to ATP phosphorylation. The results suggest that kinase-catalyzed biotinylation is well suited for phosphoproteomics studies, with particular utility towards monitoring low-abundance phosphoproteins or characterizing the influence of inhibitor drugs on protein phosphorylation.
激酶催化的蛋白质磷酸化参与了广泛的细胞事件。开发监测磷酸化的方法对于理解细胞生物学至关重要。我们实验室最近发现了激酶催化的生物素化，其中激酶利用 ATP-生物素来标记磷酸肽或磷酸化蛋白带有生物素标签。为了在细胞环境中利用激酶催化的生物素化进行磷酸蛋白的纯化和鉴定，我们对生物素标签对磷酸酶的敏感性进行了表征。我们发现肽和蛋白质底物上的磷酸化生物素基团相对不敏感于蛋白磷酸酶。为了了解磷酸酶稳定性将如何影响磷酸蛋白质组学研究应用，我们在存在激酶或磷酸酶抑制剂的情况下在细胞裂解物中进行了 ATP-生物素的激酶催化生物素化。我们发现，尽管与 ATP 磷酸化相比，生物素化对抑制剂敏感，但效果不同。结果表明，激酶催化的生物素化非常适合于磷酸蛋白质组学研究，对于监测低丰度磷酸化蛋白或表征抑制剂药物对蛋白质磷酸化的影响具有特殊的用途。
