Identification and quantification of blood–brain barrier transporters in isolated rat brain microvessels

Abstract

The blood-brain barrier (BBB) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make-up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15-fold enrichment of endothelial cell marker Glut1 mRNA, whereas markers for other cell types were not enriched. Filter-aided sample preparation was shown to be superior to in-solution sample preparation (10251 peptides vs. 7533 peptides). Label-free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP-binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.

Quantitative Proteomics of Clinically Relevant Drug-Metabolizing Enzymes and Drug Transporters and Their Intercorrelations in the Human Small Intestine

“…For protein digestion, the filter-aided sample preparation method (Wi sniewski et al,2009) was adopted as previously described (Al Feteisi et al, 2018;Al-Majdoub et al, 2019;Couto et al, 2019). Briefly, the detergent-solubilized proteins from the human total mucosal protein samples were reduced using 100 mM 1,4-dithiothreitol, followed by alkylation with 50 mM iodoacetamide.…”

GASP and FASP are Complementary for LC–MS/MS Proteomic Analysis of Drug‐Metabolizing Enzymes and Transporters in Pig Liver

“…To confirm the quality of the method, the yield of peptides in respect to the treated amount of protein can be examined, for example, using tryptophan fluorescence assay prior to LC–MS/MS analysis, although this is a relatively new approach and is far from universally used at this time. As an indicator of reproducibility and quality control in our study, the percentage identical peptides (PIP) for all combinations of technical, biological, and methodological replicates were calculated; thus low PIP between two samples means they have few common sequences . Figure B, together with the PCoA (Figure C), show that of the variance that can be explained, 65% is attributable to differences between the two methods, while only 20% due to a combination of technical and biological factors.…”

Purified IgG from aquaporin-4 neuromyelitis optica spectrum disorder patients alters blood-brain barrier permeability

“…In order to study the impact of NMO-IgG on BBB structure itself, we use an original ex vivo BBB mimicking the close relationship between NMO-IgG and brain microvessels. Fresh IBM are known to maintain in vivo BBB properties, and have been previously used for the study of endothelial molecular transporters [31,32], drug pharmacokinetic or pharmacodynamics [33], and disease pathophysiology in animal models [34,35]. However, the application of this technique on NMOSD-related models has not been previously performed.…”