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Optimization of sample preparation for transporter protein quantification in tissues by LC–MS/MS

Jankovskaja, Skaidre LU ; Kamiie, Junichi; Rezeli, Melinda LU ; Gustavsson, Lena LU ; Sugihara, Yutaka LU ; Miliotis, Tasso LU ; Ruzgas, Tautgirdas LU and Marko-Varga, György LU (2019) In Journal of Pharmaceutical and Biomedical Analysis 164. p.9-15
Abstract

Background: Reproducible quantification of drug transporter protein expression in tissues is important for predicting transporter mediated drug disposition. Many mass-spectrometry based transporter protein quantification methods result in high variability of the estimated transporter quantities. Therefore, we aimed to evaluate and optimize mass spectrometry-based quantification method for drug transporter proteins in tissues. Materials and methods: Plasma membrane (PM) proteins from mouse tissues were isolated by applying three extraction protocols: commercial plasma membrane extraction kit, tissue homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation, and PM enrichment with Tween 40.... (More)

Background: Reproducible quantification of drug transporter protein expression in tissues is important for predicting transporter mediated drug disposition. Many mass-spectrometry based transporter protein quantification methods result in high variability of the estimated transporter quantities. Therefore, we aimed to evaluate and optimize mass spectrometry-based quantification method for drug transporter proteins in tissues. Materials and methods: Plasma membrane (PM) proteins from mouse tissues were isolated by applying three extraction protocols: commercial plasma membrane extraction kit, tissue homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation, and PM enrichment with Tween 40. Moreover, five different protein digestion protocols were applied on the same PM fraction. PM isolation and digestion protocols were evaluated by measuring the amount of transporter proteins by liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. Results: Mouse liver homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation and PM enrichment with Tween 40 resulted in two times higher transporter protein quantity (Breast cancer resistance protein (Bcrp) 18.0 fmol/μg protein) in comparison with the PM samples isolated by extraction kit (Bcrp 9.8 fmol/μg protein). The evaluation of protein digestion protocols revealed that the most optimal protocol for PM protein digestion is with Lys-C and trypsin, in combination with trypsin enhancer and heat denaturation. Overall, quantities of Bcrp and Na+/K + ATPase proteins evaluated in mouse liver and kidney cortex by using our optimized PM isolation method, as well as, established digestion protocol were two to three times higher than previously reported and coefficient of variation (CV) for technical replicates was below 10%. Conclusion: We have established an improved transporter protein quantification methodology by optimizing PM isolation and protein digestion procedures. The optimized procedure resulted in a higher transporter protein yield and improved precision.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Bcrp, LC–MS/MS, Optimization, Quantification, Reproducibility, Transporter proteins
in
Journal of Pharmaceutical and Biomedical Analysis
volume
164
pages
7 pages
publisher
Elsevier
external identifiers
  • scopus:85054818718
ISSN
0731-7085
DOI
10.1016/j.jpba.2018.10.013
language
English
LU publication?
yes
id
ecf056c7-bff5-44b7-ad9c-0a2ecdfae089
date added to LUP
2018-10-26 12:37:04
date last changed
2019-02-20 11:33:32
@article{ecf056c7-bff5-44b7-ad9c-0a2ecdfae089,
  abstract     = {<p>Background: Reproducible quantification of drug transporter protein expression in tissues is important for predicting transporter mediated drug disposition. Many mass-spectrometry based transporter protein quantification methods result in high variability of the estimated transporter quantities. Therefore, we aimed to evaluate and optimize mass spectrometry-based quantification method for drug transporter proteins in tissues. Materials and methods: Plasma membrane (PM) proteins from mouse tissues were isolated by applying three extraction protocols: commercial plasma membrane extraction kit, tissue homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation, and PM enrichment with Tween 40. Moreover, five different protein digestion protocols were applied on the same PM fraction. PM isolation and digestion protocols were evaluated by measuring the amount of transporter proteins by liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. Results: Mouse liver homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation and PM enrichment with Tween 40 resulted in two times higher transporter protein quantity (Breast cancer resistance protein (Bcrp) 18.0 fmol/μg protein) in comparison with the PM samples isolated by extraction kit (Bcrp 9.8 fmol/μg protein). The evaluation of protein digestion protocols revealed that the most optimal protocol for PM protein digestion is with Lys-C and trypsin, in combination with trypsin enhancer and heat denaturation. Overall, quantities of Bcrp and Na+/K + ATPase proteins evaluated in mouse liver and kidney cortex by using our optimized PM isolation method, as well as, established digestion protocol were two to three times higher than previously reported and coefficient of variation (CV) for technical replicates was below 10%. Conclusion: We have established an improved transporter protein quantification methodology by optimizing PM isolation and protein digestion procedures. The optimized procedure resulted in a higher transporter protein yield and improved precision.</p>},
  author       = {Jankovskaja, Skaidre and Kamiie, Junichi and Rezeli, Melinda and Gustavsson, Lena and Sugihara, Yutaka and Miliotis, Tasso and Ruzgas, Tautgirdas and Marko-Varga, György},
  issn         = {0731-7085},
  keyword      = {Bcrp,LC–MS/MS,Optimization,Quantification,Reproducibility,Transporter proteins},
  language     = {eng},
  month        = {02},
  pages        = {9--15},
  publisher    = {Elsevier},
  series       = {Journal of Pharmaceutical and Biomedical Analysis},
  title        = {Optimization of sample preparation for transporter protein quantification in tissues by LC–MS/MS},
  url          = {http://dx.doi.org/10.1016/j.jpba.2018.10.013},
  volume       = {164},
  year         = {2019},
}