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

• Aquaporin-2 nephrotoxicity biomarker measurement.
• LC–MRM3 MS/MS is an alternative method to ELISA for protein quantification.
• First innovative LC–MS/MS assay for aquaporin-2 quantification.
• MRM3 improves specificity and cationic solid phase extraction reduces matrix effect.
• Absolute quantification method validation.

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 (MRM3), has recently been developed. This paper focuses on the development of an AQP2 assay in urine by LC–MS/MS, based on the MRM3 strategy, and the influence of key MRM3 parameters that enable to increase the method sensitivity by a factor of 10. Linearity is observed within the concentration range 0.5–50 ng/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.