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.
OBJECTIVE
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.


CONCLUSIONS
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.
Kamiie
Junichi
J
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
Ohtsuki
Sumio
S
Iwase
Ryo
R
Ohmine
Ken
K
Katsukura
Yuki
Y
Yanai
Kazunari
K
Sekine
Yumi
Y
Uchida
Yasuo
Y
Ito
Shingo
S
Terasaki
Tetsuya
T
D000595:Amino Acid Sequence; D000818:Animals; D001812:Blood-Brain Barrier; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D008297:Male; D026901:Membrane Transport Proteins / Q000032:analysis*; D051379:Mice; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
D026901:Membrane Transport Proteins