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

被引：418

作者：

Kamiie, Junichi ^{[1
,2
]}

Ohtsuki, Sumio ^{[1
,2
]}

Iwase, Ryo ^{[1
]}

Unine, Ken ^{[1
]}

Katsukura, Yuki ^{[1
,2
]}

Yanai, Kazunari ^{[1
]}

Sekine, Yumi ^{[1
]}

Uchida, Yasuo ^{[1
]}

Ito, Shingo ^{[1
,2
]}

Terasaki, Tetsuya ^{[1
,2
]}

机构：

[1] Tohoku Univ, Div Membrane Transport & Drug Targeting, Grad Sch Pharmaceut Sci, Aoba Ku, Sendai, Miyagi 9808578, Japan

[2] Japan Sci & Technol Agcy, SORST, Kawaguchi, Saitama, Japan

来源：

PHARMACEUTICAL RESEARCH | 2008年 / 25卷 / 06期

基金：

日本学术振兴会;

关键词：

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

D O I：

10.1007/s11095-008-9532-4

中图分类号：

O6 [化学];

学科分类号：

0703 [化学];

摘要：

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.

引用

页码：1469 / 1483

页数：15

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