LC–ESI–MS method for the monitoring of Abl 1 tyrosine kinase

A liquid chromatography–electrospray ionization–mass spectrometric (LC–ESI–MS) method was developed and validated to study Abl 1 tyrosine kinase. An online desalting system was adopted, and a transformation of the ratio of product to substrate instead of a deuterated internal standard was introduced to calculate the concentration of product. In this study, the substrate used was Abltide (KKGEAIYAAPFA-NH2). The detection was performed by selected ion monitoring (SIM) mode via positive ESI interface. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase system. The limit of quantification (LOQ) was 10 nM for the product and 25 nM for the substrate. The simple ratios of product to substrate maintained a linear relationship (R2 = 0.9997) over the ratio of 0–50% product. Intra- and inter-day precision was less than 10% and accuracy was from −1.6 to +5.3%. The validated method was applied to the Abl 1 kinase kinetic study and the Km and Vmax constants obtained for Abltide were 34.78 μM and 5.563 μmol/mg/min and for adenosine triphosphate (ATP) were 43.61 μM and 5.906 μmol/mg/min. The enzymatic reaction of Abl 1 tyrosine kinase belongs to ternary-complex mechanism.

► The kinetic constants of Abl 1 are successfully studied. ► With online desalting system, Tris–HCl buffer is used to conduct enzyme reaction. ► Enzyme reaction of Abl 1 tyrosine kinase belongs to ternary-complex mechanism.