A large-scale electrophoresis- and chromatography-based determination of gene expression profiles in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties

Abstract

Background: Brain capillary endothelial cells (BCECs) form the physiological basis of the blood-brain barrier (BBB). The barrier function is (at least in part) due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein). Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the well-established in vitro BBB model developed in our laboratory.

Results: A total of 215 protein spots (corresponding to 130 distinct proteins) were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin) and constitutes valuable evidence for predictions based on genome annotation.

Conclusions: Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets.

Figure 1

Gel image from the 2-DE of Triton X-100-extracted proteins from bovine brain capillary endothelial cells displaying the blood-brain barrier properties. 215 protein identities are reported on this 2-DE gel image and are summarized in Table S1 (see Additional File 1).

Figure 2

Cross-analysis of protein lists. The sorting and cross-analysis of protein lists was performed using nwCompare software. (A) a comparative histogram of 1D-LC analyses of the main fractions. Black areas: fraction-specific proteins; white areas: proteins found in at least three different fractions; diagonal hatching: the proportion of proteins common to the F0 and F25 fractions; vertically hatching: the proportion of proteins common to F25 and F50 fractions; horizontally hatching: the proportion of proteins common to the F50 and F75 fractions. (B) a Venn diagram showing the complementarity of the two approaches (in terms of the proteins identified).

Figure 3

Chart of biological processes and molecular functions inherent to the identified proteins. Classification of all identified proteins was performed using the Protein Analysis Through Evolutionary Relationships (PANTHER) classification system http://www.pantherdb.org. Proteins are classified by expert biologists into families and subfamilies of shared function, which are then categorized by molecular function (A) and biological process (B) ontology terms or in regard to the cellular components (C).

Figure 4

Protein-level evidence of the expression of vinculin within bovine brain capillary endothelial cells. (A) A screen print from the Mascot results page, showing the sequence coverage obtained for bovine vimentin. (B) A detailed overview of the MS/MS sequenced peptides from bovine vimentin. (C) Sequence coverage for proteins displaying similar primary amino acid sequences: MOES_BOVIN, Moesin; RADI_BOVIN, radixin; EZRI_BOVIN, ezrin (components of the ezrin-radixin-moesin (ERM) complex). The peptides common to all sequences are highlighted in blue and the amino acid sequences which are specific for a given protein are shown in red.

Figure 5

Additional evidence for the expression of vinculin within bovine brain capillary endothelial cells. (A) A screen shot from UniprotKB for bovine vinculin files. (B) A screen shot of the MASCOT results page following a search against the 57.7 UniprotKB database, showing the 12 sequenced peptides matching the human vinculin amino acid sequence. (C); A screen shot from the MASCOT results page showing the distribution of peptides over the full-length amino acid sequence of the predicted bovine vinculin (NW_001501727).