Simultaneous absolute quantitation of ATP-binding cassette transporters in normal dog tissues by signature peptide analysis using a LC/MS/MS method.

Abstract

Membrane transport proteins are fundamental components of blood-tissue barriers and affect the absorption, distribution and elimination, and interactions of many of the drugs commonly used in veterinary medicine. A quantitative, simultaneous measurement of these proteins across dog tissues is not currently available, nor is it possible with current immune-based assays such as western blot. In the present study, we aimed to develop a sensitive and specific liquid chromatography tandem-mass spectrometry (LC/MS/MS) based quantitation method that can simultaneously quantitate 14 ATP-binding cassette transporters. We applied this method to a panel of normal canine tissues and compared the LC/MS/MS results with relative messenger RNA (mRNA) abundance using quantitative real-time polymerase chain reaction (qRT-PCR). Our LC/MS/MS method is sensitive, with lower limits of quantitation ranging from 5 to 10 fmol/μg of protein. We were able to detect and/or quantitate each of the 14 transporters in at least one normal dog tissue. Relative protein and mRNA abundance within tissues did not demonstrate a significant correlation in all cases. The results presented here will provide for more accurate predictions of drug movement in dogs through incorporation into physiologically based pharmacokinetic (PBPK) models; the method described here has wide applicability to the quantitation of virtually any proteins of interest in biologic samples where validated canine antibodies do not exist.