Achour, Brahim

Al Feteisi, Hajar

Al-Majdoub, Zubida

Barber, Jill

Couto, Narciso

Rostami-Hodjegan, Amin

Journal of Neurochemistry

Hajar Al Feteisi

Zubida M. Al-Majdoub

Brahim Achour

Narciso Couto

Amin Rostami-Hodjegan

Jill Barber

Crossref

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

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 (FASP) 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 ABC and 27 SLC 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

The University of Manchester - Institutional Repository

English

Identification and Quantification of Blood-Brain Barrier Transporters in Isolated Rat Brain Microvessels

Identification and Quantification of Blood-Brain Barrier Transporters in Isolated Rat Brain Microvessels

Abstract

Similar works

Full text

Available Versions

Crossref

The University of Manchester - Institutional Repository