Secondary to the increased survival following chemotherapy, brain metastases have recently become a significant clinical problem for breast cancer patients. The aim of this study was to characterize those functional phenotypes that might enhance brain metastasis in breast cancer cells. We first analyzed by two-dimensional electrophoresis (2DE-DIGE) differences in protein expression between parental MDA-MB 435 cells and the brain metastatic variant 435-Br1, obtaining 19 identified proteins by peptide mass fingerprinting, 11 under-expressed (<2-fold) and 8 overexpressed (>2-fold) in 435-Br1. We created and analyzed protein interaction networks with a bioinformatic program (PIANA) from protein data, and it allowed us to associate 34/67-laminin receptor functionally with HSP 27, through a chaperone glucose-regulated protein GRP 94. Moreover, HSP 27 had the largest amount of direct and indirect protein interactions, forming a cluster of chaperones and cochaperones, associated through kinases to a set of intermediated filament proteins. In addition, functional groups of proteins identified were peptidase, DNA binding transcription factors, ATP synthase complex, anion transporters, and carbohydrate metabolism. Further functional analyses in cells, expression analyses in experimental tissues, and in human brain metastasis were addressed to validate the biological pathways contributing to organ-specific phenotype of brain metastasis.