Accurate and sensitive detection of dipeptidyl peptidase-IV activity by liquid chromatography with fluorescence detection
Dipeptidyl peptidase-IV (DPP-IV), important prolyl-specific peptidases in the serine hydrolase family, hydrolyzes a large number of endogenous oligopeptides. In clinical studies, the detection of DPP-IV, especially the detection of 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 the 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 by BAN obtained by detecting DPP-IV hydrolysis of GP-BAN. 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 of BAN (product of DPP-IV) as low as 5 nM (only need 2 μL), which was much lower than the others. The method also demonstrated good accuracy and precision, both variances were less than 15% between 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 in future studies.