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

Abstract

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).

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

“…On the other hand, understanding of the specific protease cleavage sites can facilitate the probability of de novo sequencing for a given protein, to predict the peptides that would result from protease digestion of the protein through an in silico digestion. Under the current practice, the combination of in silico analysis and subsequent lab experiments is widely accepted as an effective approach for developing methods to quantify membrane transporters (12,(20)(21)(22).…”

Characterization of P-Glycoprotein Humanized Mice Generated by Chromosome Engineering Technology: Its Utility for Prediction of Drug Distribution to the Brain in Humans

“…The final version may differ from this version. of the target molecules were simultaneously determined by multiplexed MRM analysis as described previously (Uchida et al, 2013;Nakamura et al, 2016). Briefly, the protein samples were denatured with 12 mM sodium deoxycholate and 12 mM N-lauroylsarcosinate and were digested by lysyl endopeptidase and trypsin after reduction and alkylation (Masuda et al, 2008).…”

“…Brain capillaries of WT, Mdr1a/1b-KO and hMDR1-MAC mice were isolated from pooled frozen brains (10 mice/group) as described previously (Uchida et al, 2013) with minor modification. Briefly, the brain homogenate was centrifuged with dextran for to concentrate brain capillaries.…”

Pharmacoproteomics-Based Reconstruction of In Vivo P-Glycoprotein Function at Blood-Brain Barrier and Brain Distribution of Substrate Verapamil in Pentylenetetrazole-Kindled Epilepsy, Spontaneous Epilepsy, and Phenytoin Treatment Models

“…Protein expression levels of 31 membrane proteins were simultaneously determined by means of multiplexed selected/multiple reaction monitoring (SRM/MRM) analysis, as described previously (Kamiie et al, 2008;Uchida et al, 2013). Isolated brain capillaries from each mouse model (50 mg protein per sample) were dissolved in denaturing buffer [500 mM Tris-HCl (pH 8.5), 7 M guanidine hydrochloride, 10 mM EDTA], which makes proteins solubilized and denatured, and then the proteins were S-carbamoylmethylated, as previously described (Kamiie et al, 2008).…”

“…For this purpose, we employed the quantitative targeted absolute proteomic (QTAP) approach to simultaneously quantify the protein expressions of many transporters (Kamiie et al, 2008;Uchida et al, 2013) in brain capillaries of normal and model mice, to identify candidate transporters potentially contributing to the change of drug permeation across the BBB in epilepsy. Among the 31 membrane proteins examined, P-gp exhibited the largest differences in protein expression levels between normal and model mice.…”