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

J Pharm Biomed Anal. 2014 Jun;94:84-91. doi: 10.1016/j.jpba.2014.01.019. Epub 2014 Jan 24.

Abstract

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 (MRM(3)) 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 MRM(3) 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 0.39ng/mL from only 1mL of urine which is compatible for endogenous level of podocin determination.

Keywords: Biomarker; MRM(3); Podocin; Protein; Quantification; Urine.

MeSH terms

  • Adult
  • Biomarkers / chemistry*
  • Biomarkers / urine*
  • Chromatography, Liquid / methods
  • Humans
  • Intracellular Signaling Peptides and Proteins / chemistry*
  • Intracellular Signaling Peptides and Proteins / urine*
  • Kidney Diseases / urine*
  • Membrane Proteins / chemistry*
  • Membrane Proteins / urine*
  • Tandem Mass Spectrometry / methods

Substances

  • Biomarkers
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • NPHS2 protein