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Table 9 Protein expression levels of transporters, receptors, claudin-5, and marker proteins in brain capillaries isolated from ddY, FVB, and C57BL/6J mice

From: A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: 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

Molecular name Protein expression level (fmol/μg protein)
ddY FVB C57BL/6J
ABC transporters    
Abcb1a (Mdr1a/P-gp) 16.4 ± 1.3c 14.2 ± 1.6e 17.8 ± 1.2
Abcc1 (Mrp1) U.L.Q. (< 0.123)c U.L.Q. (< 0.080)e U.L.Q. (< 0.121)
Abcc4 (Mrp4) 1.33 ± 0.14c 1.27 ± 0.21e 1.51 ± 0.27
Abcc5 (Mrp5) U.L.Q. (< 0.497)c U.L.Q. (< 0.412)e U.L.Q. (< 0.544)
Abcc6 (Mrp6) U.L.Q. (< 0.478)c U.L.Q. (< 0.406)e U.L.Q. (< 0.312)
Abcg2 (Bcrp) 3.74 ± 0.32c 3.21 ± 0.49e 5.48 ± 0.37a
SLC transporters    
Slc2a1 (Glut1) 82.1 ± 3.0c 90.9 ± 3.9e 101 ± 4
Slc7a5 (Lat1) 2.54 ± 1.55c 2.11 ± 0.82e 1.17 ± 0.36
Slc16a1 (Mct1) 17.3 ± 1.3c 19.9 ± 1.0e 13.7 ± 0.5b
Slc21a2 (Pgt) U.L.Q. (< 0.304) U.L.Q. (< 0.277) U.L.Q. (< 0.309)
Slc22a8 (Oat3) 1.78 ± 0.15c 1.65 ± 0.52e 2.29 ± 0.40
Slc29a4 (Pmat) U.L.Q. (< 0.220) U.L.Q. (< 0.194)e U.L.Q. (< 0.193)
Receptors    
Insr 0.738 ± 0.212d 0.637 ± 0.080e 1.13 ± 0.18
Lrp1 1.36 ± 0.42d 0.981 ± 0.072e 1.37 ± 0.33
Tfr1 4.34 ± 0.81d 3.89 ± 0.66e 5.22 ± 0.47
Tight junction protein    
Claudin-5 6.16 ± 0.20 5.50 ± 0.49e 8.07 ± 1.47
Marker proteins    
Na+/K+-ATPase 39.5 ± 1.9c 32.3 ± 1.3e 39.0 ± 0.9
γ-gtp 3.01 ± 0.47c 2.45 ± 0.12e 3.17 ± 0.36
  1. ddY and C57BL/6J mice were purchased from Japan SLC Inc., and FVB mice were purchased from CLEA Japan, Inc. Brain capillaries were freshly isolated from approximately 3 g of cerebrums pooled from 10 mice (adult, 10 weeks old, male) using a nylon mesh method. After the samples, including brain capillaries, were passed through 210- and 85-μm nylon mesh, the filtrate from the 85-μm nylon mesh was loaded onto the 20-μm nylon mesh. The brain capillaries retained on the 20-μm nylon mesh were used for absolute quantification. The recoveries of brain capillaries from ddY, FVB, and C57BL/6J mice were 110, 49.6 and 65.9 μg protein/g cerebrum, respectively. Whole tissue lysates of brain capillaries (50 μg protein) were digested with trypsin into peptide samples. The protein expression levels of the target proteins were measured by subjecting the peptide samples (30 or 3.33 μg protein) to LC-MS/MS with 500 fmol of stable isotope-labeled peptide mixture. The protein expression levels were calculated as an average of 6–8 quantitative values obtained from three or four SRM/MRM transitions in duplicate analyses. Each value represents the mean ± S.E.M. Briefly, this S.E.M. indicates the variability of the quantitative values between different transitions but does not indicate the variability between individual mice. ap < 0.001, significantly different from the protein expression level in ddY and FVB mice for bcrp (Bonferroni test). bp < 0.001, significantly different from the protein expression level in FVB mouse for mct1 (Bonferroni test). For the other molecules, there was no statistically significant difference in the protein expression levels between the three strains (p > 0.01, Bonferroni test). c, d, e These data were quantified again in the present study to eliminate experimental bias. The original results were reported in Kamiie et al. [4], Uchida et al. [6] and Agarwal et al. [23], respectively. U.L.Q., under limit of quantification.