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

Luke Anthony Wittenburg, Dominique Ramirez, Holly Conger, Daniel L. Gustafson

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)93-101
Number of pages9
JournalResearch in Veterinary Science
Volume122
DOIs
StatePublished - Feb 1 2019
Externally publishedYes

Fingerprint

ABC transporters
ATP-Binding Cassette Transporters
Dogs
peptides
Peptides
dogs
messenger RNA
Canidae
Proteins
proteins
drugs
methodology
Messenger RNA
Veterinary Medicine
Membrane Transport Proteins
transport proteins
Tandem Mass Spectrometry
Drug Interactions
Liquid Chromatography
membrane proteins

Keywords

  • ABC transporters
  • LC/MS/MS
  • Signature peptide analysis
  • Targeted proteomics

ASJC Scopus subject areas

  • veterinary(all)

Cite this

Simultaneous absolute quantitation of ATP-binding cassette transporters in normal dog tissues by signature peptide analysis using a LC/MS/MS method. / Wittenburg, Luke Anthony; Ramirez, Dominique; Conger, Holly; Gustafson, Daniel L.

In: Research in Veterinary Science, Vol. 122, 01.02.2019, p. 93-101.

Research output: Contribution to journalArticle

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