, Volume 25, Issue 6, pp 1469-1483
Date: 25 Jan 2008

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

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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.