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

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.