Optimization of liquid chromatography-multiple reaction monitoring cubed mass spectrometry assay for protein quantification: Application to aquaporin-2 water channel in human urine

摘要

Aquaporin-2 (AQP2) is a water channel protein located in the kidney collecting ducts that has been studied as a potential biomarker of a wide variety of water handling disorders and that could also be used to monitor lesions in the collecting ducts. Enzyme-linked immunosorbent assay (ELISA), the most commonly used approach for protein assay in biofluids, has a limited potential for biomarker verification due to the restricted possibility to perform multiplex assays, the cost and complexity of assay development for new candidates. Liquid chromatography tandem mass spectrometry (LC-MS/MS), in multiple reaction monitoring (MRM) mode, has been demonstrated as a powerful alternative technique, and applied to multiple protein quantification. An even more specific method, termed MRM cubed (MRM~3), has recently been developed. This paper focuses on the development of an AQP2 assay in urine by LC-MS/MS, based on the MRM~3 strategy, and the influence of key MRM~3 parameters that enable to increase the method sensitivity by a factor of 10. Linearity is observed within the concentration range 0.5-50ng/mL, intra and inter assay precision ranged from 9 to 35% at the lower limit of quantification (LLOQ), and accuracy from 94 to 114%. This assay could therefore be used in the near future to evaluate human urinary AQP2 as a potential biomarker of kidney collecting duct injury.