Absolute quantification of multidrug resistance-associated protein 2 (MRP2/ABCC2) using liquid chromatography tandem mass spectrometry

Anal Biochem. 2008 Sep 15;380(2):211-22. doi: 10.1016/j.ab.2008.05.032. Epub 2008 May 27.

Abstract

The multidrug resistance-associated protein 2 (MRP2/ABCC2) plays an important role in hepatobiliary efflux of many drugs and drug metabolites and has been reported to account for dramatic interspecies differences in the aspects of pharmacokinetics. In the present study, an absolute quantification method was developed to quantitatively measure MRP2/ABCC2 using LC-MS/MS for detection of a selective tryptic peptide. A unique 16-mer tryptic peptide was identified by conducting capillary LC nanospray ESI-Q-TOF analysis of the immunoprecipitation-enriched samples of MRP2/ABCC2 following proteolysis with trypsin. The lower limit of quantification was established to be 31.25pM with the linearity of the standard curve spanned to 2500pM. Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%. Using this method, we successfully determined the absolute amount of MRP2/ABCC2 protein in MRP2/ABCC2 gene-transfected MDCK cells as well as the basal levels of canine Mrp2/Abcc2 protein in MDCK cells. Our findings also demonstrate that the sensitivity of this method exceeds the sensitivity of immunoblotting assay which was not able to detect the basal levels of canine Mrp2/Abcc2 in MDCK cells. The method could be directly applicable to many current research needs related to MRP2/ABCC2 protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Calibration
  • Cell Line
  • Chromatography, Liquid / methods*
  • Dogs
  • Humans
  • Immunoprecipitation
  • Molecular Sequence Data
  • Multidrug Resistance-Associated Proteins / analysis*
  • Multidrug Resistance-Associated Proteins / biosynthesis
  • Multidrug Resistance-Associated Proteins / genetics
  • Nanotechnology / methods
  • Recombinant Proteins / analysis
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Reproducibility of Results
  • Sequence Alignment
  • Spectrometry, Mass, Electrospray Ionization / methods
  • Tandem Mass Spectrometry / methods*

Substances

  • Multidrug Resistance-Associated Proteins
  • Recombinant Proteins
  • multidrug resistance-associated protein 2