Background: Breast cancer is worldwide the second most common type of cancer after lung cancer. Plasma proteome profiling may have a higher chance to identify protein changes between plasma samples such as normal and breast cancer tissues. Breast cancer cell lines have long been used by researches as model system for identifying protein biomarkers. A comparison of the set of proteins which change in plasma with previously published findings from proteomic analysis of human breast cancer cell lines may identify with a higher confidence a subset of candidate protein biomarker.Results: In this study, we analyzed a liquid chromatography (LC) coupled tandem mass spectrometry (MS/MS) proteomics dataset from plasma samples of 40 healthy women and 40 women diagnosed with breast cancer. Using a two-sample t-statistics and permutation procedure, we identified 254 statistically significant, differentially expressed proteins, among which 208 are over-expressed and 46 are under-expressed in breast cancer plasma. We validated this result against previously published proteomic results of human breast cancer cell lines and signaling pathways to derive 25 candidate protein biomarkers in a panel. Using the pathway analysis, we observed that the 25 " activated" plasma proteins were present in several cancer pathways, including 'Complement and coagulation cascades', 'Regulation of actin cytoskeleton', and 'Focal adhesion', and match well with previously reported studies. Additional gene ontology analysis of the 25 proteins also showed that cellular metabolic process and response to external stimulus (especially proteolysis and acute inflammatory response) were enriched functional annotations of the proteins identified in the breast cancer plasma samples. By cross-validation using two additional proteomics studies, we obtained 86% and 83% similarities in pathway-protein matrix between the first study and the two testing studies, which is much better than the similarity we measured with proteins.Conclusions: We presented a 'systems biology' method to identify, characterize, analyze and validate panel biomarkers in breast cancer proteomics data, which includes 1) t statistics and permutation process, 2) network, pathway and function annotation analysis, and 3) cross-validation of multiple studies. Our results showed that the systems biology approach is essential to the understanding molecular mechanisms of panel protein biomarkers.