CEA, DSV, IBEB, SBTN, Bagnols-sur-Cèze F-30207, France.
Anal Chem. 2012 Nov 20;84(22):9963-70. doi: 10.1021/ac3021505. Epub 2012 Oct 30.
Protein phosphorylation is a critical signaling mechanism in cellular regulation and stress response, and more than 95% of the phosphorylations are targeted toward Ser or Thr amino-acid residues. The classical techniques for analyzing phospho-amino acid residues use radioisotopes or sequence-specific antibodies. However, both practical and economical limitations have prevented their development, and we here propose an original approach for the detection of phospho-Ser/Thr residues. It requires no antibody and exploits the patented homogeneous time-resolved fluorescence (HTRF) technology, in association with a 3-step chemical transformation of phospho-amino acids into fluorescent derivatives. The process involves: (i) alkaline β-elimination of the phosphorylated group, (ii) Michael addition of a bifunctional group, and then (iii) introduction of cyanin-5 as fluorescent acceptor for HTRF. The donor fluorescent moiety at the N-terminus of the phosphorylated peptide is a streptavidin europium cryptate conjugate. After its development, the detection system has been validated on synthetic peptide substrates of Chk2, a key protein kinase activated in response to DNA damage and involved in cell cycle arrest. The results showed a good correlation with known specificity profiles. Interestingly, the detection system is versatile, easy to implement, and suitable for multiple parallel analyses.
蛋白质磷酸化是细胞调节和应激反应中的一个关键信号机制，超过 95%的磷酸化靶向丝氨酸或苏氨酸氨基酸残基。分析磷酸化氨基酸残基的经典技术使用放射性同位素或序列特异性抗体。然而，实际和经济上的限制阻碍了它们的发展，我们在这里提出了一种用于检测磷酸丝氨酸/苏氨酸残基的新方法。它不需要抗体，利用了专利的均相时间分辨荧光（HTRF）技术，结合了磷酸氨基酸转化为荧光衍生物的 3 步化学转化。该过程包括：（i）磷酸基团的碱性β消除，（ii）双功能基团的迈克尔加成，然后（iii）将青色素 5 作为荧光受体引入 HTRF。磷酸肽 N 端的供体荧光部分是链霉亲和素铕 cryptate 缀合物。该检测系统开发后，已在 Chk2 的合成肽底物上进行了验证，Chk2 是一种在 DNA 损伤响应中被激活的关键蛋白激酶，参与细胞周期停滞。结果与已知的特异性谱相关性良好。有趣的是，该检测系统具有通用性、易于实施，并且适用于多种并行分析。
