Pharmaceutical Research

, Volume 25, Issue 6, pp 1469–1483 | Cite as

Quantitative Atlas of Membrane Transporter Proteins: Development and Application of a Highly Sensitive Simultaneous LC/MS/MS Method Combined with Novel In-silico Peptide Selection Criteria

  • Junichi Kamiie
  • Sumio Ohtsuki
  • Ryo Iwase
  • Ken Ohmine
  • Yuki Katsukura
  • Kazunari Yanai
  • Yumi Sekine
  • Yasuo Uchida
  • Shingo Ito
  • Tetsuya Terasaki
Research Paper

Abstract

Purpose

To develop an absolute quantification method for membrane proteins, and to construct a quantitative atlas of membrane transporter proteins in the blood–brain barrier, liver and kidney of mouse.

Methods

Mouse tissues were digested with trypsin, and mixed with stable isotope labeled-peptide as a quantitative standard. The amounts of transporter proteins were simultaneously determined by liquid chromatography–tandem mass spectrometer (LC/MS/MS).

Results

The target proteins were digested in-silico, and target peptides for analysis were chosen on the basis of the selection criteria. All of the peptides selected exhibited a detection limit of 10 fmol and linearity over at least two orders of magnitude in the calibration curve for LC/MS/MS analysis. The method was applied to obtain the expression levels of 34 transporters in liver, kidney and blood–brain barrier of mouse. The quantitative values of transporter proteins showed an excellent correlation with the values obtained with existing methods using antibodies or binding molecules.

Conclusion

A sensitive and simultaneous quantification method was developed for membrane proteins. By using this method, we constructed a quantitative atlas of membrane transporter proteins at the blood–brain barrier, liver and kidney in mouse. This technology is expected to have major implications for various fields of biomedical science.

Key words

ABC transporter LC/MS/MS multiple reaction monitoring (MRM) pharmacoproteomics SLC transporter 

Abbreviations

ABC transporters

ATP binding cassette transporters

CV

coefficient of variation

ESI

electro-spray ionization

HSA

human serum albumin

HUGO

Human Genome Organization

LC

liquid chromatography

MRM

multiple reaction monitoring

MS/MS

tandem mass spectrometry

PBS

phosphate-buffered saline

PMSF

phenylmethylsulfonyl fluoride

Q1

quadrupole 1

Q3

quadrupole 3

SLC transporters

solute carrier family of transporters

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Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Junichi Kamiie
    • 1
    • 2
  • Sumio Ohtsuki
    • 1
    • 2
  • Ryo Iwase
    • 1
  • Ken Ohmine
    • 1
  • Yuki Katsukura
    • 1
    • 2
  • Kazunari Yanai
    • 1
  • Yumi Sekine
    • 1
  • Yasuo Uchida
    • 1
  • Shingo Ito
    • 1
    • 2
  • Tetsuya Terasaki
    • 1
    • 2
  1. 1.Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical SciencesTohoku UniversitySendaiJapan
  2. 2.SORST of the Japan Science and Technology AgencyKawaguchiJapan

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