Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190.
Analyst. 2010 Nov;135(11):2858-63. doi: 10.1039/c0an00339e. Epub 2010 Sep 13.
A MALDI-TOF mass spectrometric method for rapid screening of protein tyrosine kinase (PTK) inhibitors has been developed. To circumvent the ion suppression of phosphorylated substrate peptides caused by the presence of high abundant non-phosphorylated peptides in the enzymatic reaction mixtures, a separation and enrichment process of the phosphorylated peptides from complex mixtures was carried out by using an in-house fabricated TiO(2) nanoparticle-coated capillary column prior to the MS analysis. With a synthetic phosphopeptide (DAIpYAAPFAKKK), of which the sequence is similar to that of the substrate (EAIYAAPFAKKK) of the Abelson tyrosine kinase (Abl), as the internal standard, the signal ratio of the phosphorylated substrate to the standard detected by MALDI-TOF MS is linearly correlated with the molar ratio of the two phosphopeptides over the range of 0.3 to 3 with r(2) = 0.99. We validated the MS method by determining the IC(50) value of imatinib, an Abl inhibitor for clinical treatment of chronic myelogenous leukaemia (CML). The obtained IC(50) value (234 nM) is consistent with that determined by ELISA (291 nM). Then, six analogues of imatinib synthesized in our laboratory were screened using the method, giving rise to inhibitory potential results which are in good agreement with the docking analysis data. The developed method is sensitive, operationally simple, does not require isotope-labelling and is cost/time effective, providing an alterative method for rapid screening of PTK inhibitors as therapeutic agents for tumours.
建立了一种基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）快速筛选蛋白酪氨酸激酶（PTK）抑制剂的方法。为了避免由于酶反应混合物中存在大量非磷酸化肽而导致磷酸化底物肽的离子抑制，通过使用自制的 TiO(2)纳米粒子涂覆的毛细管柱，在 MS 分析之前，从复杂混合物中分离和富集磷酸化肽。用一种与 Abelson 酪氨酸激酶（Abl）的底物（EAIYAAPFAKKK）序列相似的合成磷酸肽（DAIpYAAPFAKKK）作为内标，通过 MALDI-TOF MS 检测到的磷酸化底物与标准的信号比与两种磷酸肽的摩尔比在 0.3 到 3 范围内呈线性相关，r(2) = 0.99。我们通过测定伊马替尼（一种用于治疗慢性髓性白血病（CML）的 Abl 抑制剂）的 IC(50)值验证了该 MS 方法。得到的 IC(50)值（234 nM）与 ELISA 法（291 nM）测定的结果一致。然后，使用该方法筛选了我们实验室合成的 6 种伊马替尼类似物，得到的抑制潜力结果与对接分析数据非常吻合。该方法灵敏、操作简单，不需要同位素标记，成本/时间效率高，为快速筛选肿瘤治疗用的 PTK 抑制剂提供了一种替代方法。
