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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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.
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).
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.
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 wordsABC transporter LC/MS/MS multiple reaction monitoring (MRM) pharmacoproteomics SLC transporter
- ABC transporters
ATP binding cassette transporters
coefficient of variation
human serum albumin
Human Genome Organization
multiple reaction monitoring
tandem mass spectrometry
- SLC transporters
solute carrier family of transporters
We would like to thank Ms N. Funayama for secretarial assistance. This study was supported in part by a Grant-in-Aid for Young Scientist (B) and Scientific Research on Priority Areas (17081002) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Grant-in-Aid for Scientific Research (S), Scientific Research (B) and a 21st Century Center of Excellence (COE) Program grant from the Japan Society for the Promotion of Science. This study was also supported in part by the Industrial Technology Research Grant Program from New Energy and the Industrial Technology Development Organization (NEDO) of Japan.
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