Development\nof a Multiplexed LC-MS/MS Assay for the\nQuantitation of Podocyte Injury Biomarkers Nephrin, Podocalyxin, and\nPodocin in Human Urine

CKD is frequently diagnosed only after a significant\nprogression.\nGFR is the most common indicator of kidney function but is limited\nin detecting early CKD cases and distinguishing glomerular, tubular,\nand global CKD. Aiming to provide a glomeruli specific biomarker assay,\nwe developed a peptide immunoaffinity targeted mass spectrometry method\nfor the quantitation of three podocyte specific proteins in human\nurine: nephrin, podocalyxin, and podocin. Proteins in urine were precipitated,\nstable isotope labeled peptide standards incorporated, and digested\nwith trypsin. Target peptides were enriched using an online antibody\ncolumn prior to LC-MS/MS. The performance metrics for nephrin, podocalyxin,\nand podocin were evaluated: The lower limits of quantitation were\n3.8, 22.0, and 5.4 pM, respectively. The intraplate relative error\n(RE) was within ±10.6%, ± 10.4%, and ±16.1%, and coefficient\nof variation (CV) was ≤27.2%, ≤ 14.1%, and ≤20.7%\naccordingly. The interplate RE was within ±7.0%, ± 3.8%,\nand ±3.0%, and CV was ≤17.2%, ≤ 12.1%, and ≤20.0%\nfor the three analytes. The urinary nephrin, podocalyxin, and podocin\nconcentrations in 60 healthy volunteers and 20 disease samples was\nmeasured, thereby establishing the basal levels of these protein and\nenabling future evaluation of their roles as noninvasive biomarkers\nof glomerular injury in the clinic.