TY - JOUR
T1 - A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS
T2 - Application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood-brain barrier in ddY, FVB, and C57BL/6J mice
AU - Uchida, Yasuo
AU - Tachikawa, Masanori
AU - Obuchi, Wataru
AU - Hoshi, Yutaro
AU - Tomioka, Yusuke
AU - Ohtsuki, Sumio
AU - Terasaki, Tetsuya
N1 - Funding Information:
We thank Ms. N. Handa and A. Niitomi for secretarial assistance. The studies were supported in part by four Grants-in-Aid for Scientific Research (S) [KAKENHI: 18109002], Scientific Research (A) [KAKENHI: 24249011], Young Scientists (B) [KAKENHI: 23790170], and the Japan Society for the Promotion of Science (JSPS) Fellows [KAKENHI: 207291] from the JSPS. The studies were also supported in part by a Grant-in-Aid for Scientific Research on Priority Area [KAKENHI: 17081002] from The Ministry of Education, Culture, Sports, Science and Technology (MEXT), 3 Grants for the Development of Creative Technology Seeds Supporting Program for Creating University Ventures, the Creation of Strategic Innovation Project, and the Revitalization Promotion Program (A-STEP) from the Japan Science and Technology Agency (JST). Furthermore, the studies were also supported in part by an Industrial Technology Research Grant Program from New Energy and the Industrial Technology Development Organization (NEDO) of Japan, a Health and Labour Sciences Research Grant from The Ministry of Health, Labour and Welfare, the Funding Program for Next Generation World-Leading Researchers by the Cabinet Office, Government of Japan, and Proteomedix Frontiers Co. Ltd.
PY - 2013/6/8
Y1 - 2013/6/8
N2 - Proteomics has opened a new horizon in biological sciences. Global proteomic analysis is a promising technology for the discovery of thousands of proteins, post-translational modifications, polymorphisms, and molecular interactions in a variety of biological systems. The activities and roles of the identified proteins must also be elucidated, but this is complicated by the inability of conventional proteomic methods to yield quantitative information for protein expression. Thus, a variety of biological systems remain " black boxes" Quantitative targeted absolute proteomics (QTAP) enables the determination of absolute expression levels (mol) of any target protein, including low-abundance functional proteins, such as transporters and receptors. Therefore, QTAP will be useful for understanding the activities and roles of individual proteins and their differences, including normal/disease, human/animal, or in vitro/in vivo. Here, we describe the study protocols and precautions for QTAP experiments including in silico target peptide selection, determination of peptide concentration by amino acid analysis, setup of selected/multiple reaction monitoring (SRM/MRM) analysis in liquid chromatography-tandem mass spectrometry, preparation of protein samples (brain capillaries and plasma membrane fractions) followed by the preparation of peptide samples, simultaneous absolute quantification of target proteins by SRM/MRM analysis, data analysis, and troubleshooting. An application of QTAP in biological sciences was introduced that utilizes data from inter-strain differences in the protein expression levels of transporters, receptors, tight junction proteins and marker proteins at the blood-brain barrier in ddY, FVB, and C57BL/6J mice. Among 18 molecules, 13 (abcb1a/mdr1a/P-gp, abcc4/mrp4, abcg2/bcrp, slc2a1/glut1, slc7a5/lat1, slc16a1/mct1, slc22a8/oat3, insr, lrp1, tfr1, claudin-5, Na+/K+-ATPase, and γ-gtp) were detected in the isolated brain capillaries, and their protein expression levels were within a range of 0.637-101 fmol/μg protein. The largest difference in the levels between the three strains was 2.2-fold for 13 molecules, although bcrp and mct1 displayed statistically significant differences between C57BL/6J and the other strain(s). Highly sensitive simultaneous absolute quantification achieved by QTAP will increase the usefulness of proteomics in biological sciences and is expected to advance the new research field of pharmacoproteomics (PPx).
AB - Proteomics has opened a new horizon in biological sciences. Global proteomic analysis is a promising technology for the discovery of thousands of proteins, post-translational modifications, polymorphisms, and molecular interactions in a variety of biological systems. The activities and roles of the identified proteins must also be elucidated, but this is complicated by the inability of conventional proteomic methods to yield quantitative information for protein expression. Thus, a variety of biological systems remain " black boxes" Quantitative targeted absolute proteomics (QTAP) enables the determination of absolute expression levels (mol) of any target protein, including low-abundance functional proteins, such as transporters and receptors. Therefore, QTAP will be useful for understanding the activities and roles of individual proteins and their differences, including normal/disease, human/animal, or in vitro/in vivo. Here, we describe the study protocols and precautions for QTAP experiments including in silico target peptide selection, determination of peptide concentration by amino acid analysis, setup of selected/multiple reaction monitoring (SRM/MRM) analysis in liquid chromatography-tandem mass spectrometry, preparation of protein samples (brain capillaries and plasma membrane fractions) followed by the preparation of peptide samples, simultaneous absolute quantification of target proteins by SRM/MRM analysis, data analysis, and troubleshooting. An application of QTAP in biological sciences was introduced that utilizes data from inter-strain differences in the protein expression levels of transporters, receptors, tight junction proteins and marker proteins at the blood-brain barrier in ddY, FVB, and C57BL/6J mice. Among 18 molecules, 13 (abcb1a/mdr1a/P-gp, abcc4/mrp4, abcg2/bcrp, slc2a1/glut1, slc7a5/lat1, slc16a1/mct1, slc22a8/oat3, insr, lrp1, tfr1, claudin-5, Na+/K+-ATPase, and γ-gtp) were detected in the isolated brain capillaries, and their protein expression levels were within a range of 0.637-101 fmol/μg protein. The largest difference in the levels between the three strains was 2.2-fold for 13 molecules, although bcrp and mct1 displayed statistically significant differences between C57BL/6J and the other strain(s). Highly sensitive simultaneous absolute quantification achieved by QTAP will increase the usefulness of proteomics in biological sciences and is expected to advance the new research field of pharmacoproteomics (PPx).
KW - Absolute expression level
KW - Blood-brain barrier
KW - In silico peptide selection criteria
KW - LC-MS/MS
KW - Pharmacoproteomics (PPx)
KW - Quantitative targeted absolute proteomics (QTAP)
KW - Receptor
KW - Strain difference
KW - Tight junction protein
KW - Transporter
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U2 - 10.1186/2045-8118-10-21
DO - 10.1186/2045-8118-10-21
M3 - Article
C2 - 23758935
AN - SCOPUS:84878654332
VL - 10
JO - Fluids and Barriers of the CNS
JF - Fluids and Barriers of the CNS
SN - 2045-8118
IS - 1
M1 - 21
ER -