Absolute quantification of podocalyxin, a potential biomarker of glomerular injury in human urine, by liquid chromatography–mass spectrometry

• A quantification method of human podocalyxin in urine by LC–MS/MS has been developed.
• Inter assay precisions and accuracies are <10% and <20% respectively.
• The lower limit of quantification is 0.78 ng/mL from 1 mL of urine.
• The quantification method is compatible with endogenous level of podocalyxin.

Podocalyxin is a protein present in specialized glomerulus cells called podocytes and may be released in the urine in case of kidney injury. In this context, its quantification could be of great interest in order to monitor glomerular injury. Liquid chromatography tandem mass spectrometry (LC–MS/MS), in selected reaction monitoring (SRM) mode, has been demonstrated as a powerful technique that can be applied to protein quantification. This paper describes the development of a quantification method of human podocalyxin in urine by LC–MS/MS in SRM mode by monitoring one proteotypic peptide with an isotope-dilution standardization strategy employing 13C/15N labelled peptides. Inter/intra assay precisions and accuracies of the assay were below 10% and between 90% and 106.1%, respectively. In addition, the method was linear between 0.78 and 100 ng/mL and could therefore be used to quantify endogenous level of podocalyxin that was estimated between 15.2 and 44.2 ng/mL in urine samples from healthy donor.