A differential proteomic approach identifies structural and functional components that contribute to the differentiation of brain capillary endothelial cells Gwënaël Pottiez a, b, c , 1 , Sophie Duban-Deweer a, b, c , 1 , Barbara Deracinois a, b, c , Fabien Gosselet a, b, c , Luc Camoin d, e , Johan Hachani a, b, c , Pierre-Olivier Couraud d, e , Roméo Cecchelli a, b, c , Marie-Pierre Dehouck a, b, c , Laurence Fenart a, b, c , Yannis Karamanos a, b, c , Christophe Flahaut a, b, c , ⁎ a Univ Lille Nord de France, F-59000 Lille, France b UArtois, LBHE, F-62307 Lens, France c IMPRT-IFR114, F-59000, Lille, France d Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France e INSERM, U567, Paris, France ARTICLE INFO ABSTRACT Article history: Received 25 April 2011 Accepted 1 September 2011 Available online 23 September 2011 When in the vicinity of astrocytes, brain capillary endothelial cells (BCECs) develop the characteristic structural and functional features of the blood-brain barrier (BBB). The latter has low cellular permeability and restricts various compounds from entering the brain. We recently reported that the cytoskeleton-related proteins actin, gelsolin and filamin-A undergo the largest quantitative changes in bovine BCECs after re-induction of BBB functions by co- culture with glial cells. In the present study, we used an in-depth, proteomic approach to quantitatively compare differences in Triton-X-100-solubilized proteins from bovine BCECs with limited or re-induced BBB functions (i.e. cultured in the absence or presence of glial cells, respectively). The 81 protein spots of differing abundance were linked to 55 distinct genes. According to the Protein ANalysis THrough Evolutionary Relationships classification system and an Ingenuity Pathway Analysis, these quantitative changes mainly affected pro- teins involved in (i) cell structure and motility and (ii) protein metabolism and modification. The fold-changes affecting HSPB1, moesin and ANXA5 protein levels were confirmed by west- ern blot analysis but were not accompanied by changes in the corresponding mRNA expression Keywords: Proteomics Blood-brain barrier Cytoskeletal remodelling Triton X-100 soluble proteins JOURNAL OF PROTEOMICS 75 (2011) 628 – 641 Abbreviations: 1D-LC, one-dimensional liquid chromatography; 2-DE, two-dimensional electrophoresis; BBB, blood-brain barrier; BCEC, brain capillary endothelial cell; CLIC, chloride intracellular channel; ERM, ezrin/radixin/moesin; HSP, heat-shock protein; IPA, Ingenuity Pathway Analysis; PDI, protein disulphide isomerase; Pe, permeability coefficient; PMF, peptide mass fingerprinting; TSFs, Triton-X- 100-solubilized fractions. ⁎ Corresponding author at: Laboratoire de physiopathologie de la barrière hémato-encéphalique, Université d'Artois, Faculté des Sciences Jean Perrin, rue Jean Souvraz, S.P. 18, F-62307 Lens cedex, France. Tel.: +33 3 21 79 17 80; fax: +33 3 21 79 17 36. E-mail addresses: gwenael.pottiez@gmail.com (G. Pottiez), sophie.duban@univ-artois.fr (S. Duban-Deweer), barbara.deracinois@univ-artois.fr (B. Deracinois), fabien.gosselet@univ-artois.fr (F. Gosselet), luc.camoin@inserm.fr (L. Camoin), Johan.hachani@univ-artois.fr (J. Hachani), pierre-olivier.couraud@inserm.fr (P.-O. Couraud), romeo.cecchelli@univ-artois.fr (R. Cecchelli), mpierre.dehouck@univ-artois.fr (M.-P. Dehouck), laurence.tilloy@univ-artois.fr (L. Fenart), yannis.karamanos@univ-artois.fr (Y. Karamanos), christophe.flahaut@univ-artois.fr (C. Flahaut). 1 GP and SDD equally contributed to this study. 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.09.002 Available online at www.sciencedirect.com www.elsevier.com/locate/jprot