Absolute quantification of podocin, a potential biomarker of glomerular injury in human urine, by liquid chromatography–multiple reaction monitoring cubed mass spectrometry

• We develop an innovative LC–MRM3-MS/MS assay for podocin quantification from 1 mL of urine.
• LC–MRM3-MS/MS is an alternative method to ELISA for protein quantification.
• MRM3 and cationic solid phase extraction improve specificity and sensitivity without immuno-depletion of major proteins.
• Method was validated in absolute quantification.

Glomeruli play a major role in the kidney function since they are involved in primary urine formation. It is then crucial to dispose of methods to monitor glomerular injury, especially in drug development. In this context, quantification of podocin could be of great interest since it is a protein exclusively present in highly specialized glomerulus cells called podocytes. Immunoassays are the most commonly used approach for protein assays. However, they rely on the availability of specific antibodies. When such antibodies are not available, liquid chromatography tandem mass spectrometry (LC–MS/MS), in selected reaction monitoring (SRM) or in multiple reaction monitoring cubed (MRM3) mode, has been demonstrated as a powerful alternative technique, and can be applied to multiple protein quantification. This paper describes the development of a quantification method of human podocin in urine by LC–MS/MS in MRM3 mode. Inter assay precision and accuracy ranged from 7 to 20% and from 105 to 112% respectively and the lower limit of quantification (LLOQ) was 0.39 ng/mL from only 1 mL of urine which is compatible for endogenous level of podocin determination.

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