The subsequent mass spectrometry analysis was used to identify the peptides digested from these spots individually picked up from 2D gels. The additional advantage of this experimental approach is that 2D separation greatly reduces the complexity of the samples undergoing subsequent MS analysis and diminishes a possibility of the suppression of MS identification of low abundance peptides by highly abundant proteins. We followed by the Ingenuity Pathway Analysis to ascertain major signaling networks associated with these proteins and hence potentially involved in breast cancer metastasis to the brain as well as to compare them with signaling networks associated with a list of 19 brain metastasis associated proteins differentially expressed in brain-targeting 435-Br1 cells compared with parental MDA-MB-435 cells identified recently by the group of Dr. Angels Sierra using a similar approach, or a 17-gene classifier associated with breast cancer brain relapse published recently by the group of Dr. Joan Massague based on a microarray analysis of Niltubacin clinically annotated breast tumors from 368 patients. Our results demonstrated that, even though only 2 out of 31 proteins were in common between the MDA-MB-231- based set identified by our group in this study and MB-435-based set, and none of these proteins were present in a 17-gene breast cancer brain relapse signature, they all converged on the major signaling networks involving TNFa/TGFb-, NFkB-, HSP70-, TP53, and IFNa/c-related pathways. These findings, showing that different experimental systems and approaches yield highly related signaling networks associated with the differentially expressed proteins and/or genes, suggest strongly that these signaling networks could be essential for a successful colonization of the brain by metastatic breast carcinoma cells. Recently, using a similar approach, the group of Dr. Angels Sierra identified a set of 19 proteins differentially expressed in brain-targeting 435-Br1 cells compared with parental MDA-MB-435 cells. Between the two sets of brain-metastasis-associated proteins only 2 out of 31 proteins, HSPA8 and vimentin, were the same. Thus, we carried out the hypergeometric distribution test. The results of this analysis demonstrated that it is highly unlikely that the 2-protein overlap found between the two sets occurred by chance. Next, to reveal the key signaling pathways or networks related to the set of brain metastasis-associated proteins identified by the group of Dr. Sierra using MDA-MB-435-based experimental system, we imported the list of these 19 proteins into the IPA software. The IPA analysis revealed 3 signaling networks associated with this set of proteins. Network 1 included 12 out of 19 differentially expressed proteins and involved TNFa/TGFb-, NFKB-, HSP-70-, and MAPK-associated major signaling pathways.