Accurate and sensitive detection of dipeptidyl peptidase-IV activity by liquid chromatography with fluorescence detection

Abstract

Dipeptidyl peptidase-IV (DPP-IV), an important prolyl-specific peptidase in the serine hydrolase family, hydrolyzes a large number of endogenous oligopeptides. In clinical studies, the detection of DPP-IV, especially the detection of its activity, is particularly important. We previously reported a specific fluorescent two-photon probe for DPP-IV. In this study, to further verify that our probe can be used for DPP-IV activity detection, we developed an accurate and sensitive method for the determination of DPP-IV activity in human tissue microsomes, cell homogenates or cell supernatants by liquid chromatography-fluorescence detection (LC-FD). This method is based on the fluorescence generated from BAN obtained from the hydrolysis of GP-BAN by detecting DPP-IV. The linearity, sensitivity, recovery, precision and stability of the method were fully validated. The method based on LC-FD has a lower limit of quantitation for BAN (product of DPP-IV), as low as 5 nM (only needs 2 μL), which is much lower than that of the others. This method also demonstrated good accuracy and precision; variances of both were less than 15% between the intra- and inter-assay. In addition, the method was successfully applied to the determination of DPP-IV activity in various cell homogenates and cell supernatants. The development of the LC-FD method will help to understand the expression and function of DPP-IV in different biological samples in humans for future studies.