Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences vol:897 pages:17-21
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 C-18 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 (R-2 = 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 K-m and V-max constants obtained for Abltide were 34.78 mu M and 5.563 mu mol/mg/min and for adenosine triphosphate (ATP) were 43.61 mu M and 5.906 mu mol/mg/min. The enzymatic reaction of Abl 1 tyrosine kinase belongs to ternary-complex mechanism. (C) 2012 Elsevier B.V. All rights reserved.