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 language | English (US) |
|---|---|
| Pages (from-to) | 93-101 |
| Number of pages | 9 |
| Journal | Research in Veterinary Science |
| Volume | 122 |
| DOIs | |
| State | Published - Feb 1 2019 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - Simultaneous absolute quantitation of ATP-binding cassette transporters in normal dog tissues by signature peptide analysis using a LC/MS/MS method
AU - Wittenburg, Luke Anthony
AU - Ramirez, Dominique
AU - Conger, Holly
AU - Gustafson, Daniel L.
PY - 2019/2/1
Y1 - 2019/2/1
N2 - 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.
AB - 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.
KW - ABC transporters
KW - LC/MS/MS
KW - Signature peptide analysis
KW - Targeted proteomics
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UR - http://www.scopus.com/inward/citedby.url?scp=85057123898&partnerID=8YFLogxK
U2 - 10.1016/j.rvsc.2018.11.009
DO - 10.1016/j.rvsc.2018.11.009
M3 - Article
VL - 122
SP - 93
EP - 101
JO - Research in Veterinary Science
JF - Research in Veterinary Science
SN - 0034-5288
ER -