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).

Background: Organic anion transporting polypeptide (Oatp) transporters at the blood–brain barrier (BBB) and the blood-retinal barrier (BRB), which consists of retinal capillary endothelial cells and retinal pigment epithelial cells, are major determinants of the control of anionic drugs into the brain and retina. Although Oatp1a4 (Slco1a4) and Oatp1c1 (Slco1c1) are known to be expressed in the abluminal and luminal membrane of the rat BBB and Oatp1a4 is known to be expressed at the BRB, the expression and localization of Oatp1c1 at the BRB and subcellular localization of Oatp1a4 at the BRB have received little attention. Therefore, the purpose of present study was to determine the cellular and subcellular localization of Oatp1a4 and 1c1 at the BRB. Methods: We used guinea pig polyclonal antibodies to Oatp1a4 and 1c1 for immunoblot and immunohistochemical analysis to determine their cellular and subcellular distributions in the rat retina. We compared these distributions with those of the glucose transporter 1 (GLUT1/Slc2a1). Whole brain, brain capillary fractions and kidney were used as control. Results: Oatp1a4 and 1c1 immunoreactivities were detected in the rat retinal capillaries and co-localized with GLUT1, suggesting that both proteins are located on the abluminal and luminal membrane of the retinal capillary endothelial cells. Oatp1a4 and 1c1 immunoreactivities were preferentially detected on the apical and basolateral membrane of rat retinal pigment epithelial cells, respectively, suggesting that Oatp1a4 and 1c1 are localized on the apical membrane and the basolateral membrane of the retinal pigment epithelial cells, respectively. Conclusion: Oatp1a4 and 1c1 are present at the BRB and contribute to the transcellular transport of amphipathic organic anions across the BRB.

Background: Peripheral administration of lipopolysaccharide (LPS) induces inflammation and increases cerebral prostaglandin E₂(PGE₂) concentration. PGE₂is eliminated from brain across the blood-brain barrier (BBB) in mice, and this process is inhibited by intracerebral or intravenous pre-administration of anti-inflammatory drugs and antibiotics such as cefmetazole and cefazolin that inhibit multidrug resistance-associated protein 4 (Mrp4/Abcc4)-mediated PGE₂transport. The purpose of this study was to examine the effect of LPS-induced inflammation on PGE₂elimination from brain, and whether antibiotics further inhibit PGE₂elimination in LPS-treated mice.Methods: [³H]PGE₂elimination across the BBB of intraperitoneally LPS-treated mice was assessed by the brain efflux index (BEI) method. Transporter protein amounts in brain capillaries were quantified by liquid chromatography-tandem mass spectrometry.Results: The apparent elimination rate of [³H]PGE₂from brain was lower by 87%, in LPS-treated mice compared with saline-treated mice. The Mrp4 protein amount was unchanged in brain capillaries of LPS-treated mice compared with saline-treated mice, while the protein amounts of organic anion transporter 3 (Oat3/Slc22a8) and organic anion transporting polypeptide 1a4 (Oatp1a4/Slco1a4) were decreased by 26% and 39%, respectively. Either intracerebral or intravenous pre-administration of cefmetazole further inhibited PGE₂elimination in LPS-treated mice. However, intracerebral or intravenous pre-administration of cefazolin had little effect on PGE₂elimination in LPS-treated mice, or in LPS-untreated mice given Oat3 and Oatp1a4 inhibitors. These results indicate that peripheral administration of cefmetazole inhibits PGE₂elimination across the BBB in LPS-treated mice.Conclusion: PGE₂elimination across the BBB is attenuated in an LPS-induced mouse model of inflammation. Peripheral administration of cefmetazole further inhibits PGE₂elimination in LPS-treated mice.