Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix- assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS), incorporated with online database searching, were per- formed to investigate differential proteins of breast cancer and adjacent normal breast tissues. Considering that serum albumin is abundantly presented in nor- mal control samples, 15 differential spots detected in 11 out of 12 (91.7%) breast cancer samples were identified by online SIENA-2DPAGE database searching and MALDI-TOF/TOF-MS analysis. The results indicate that pathological changes of breast cancer are concerned with augmentation of substance metabolism, promo- tion of proteolytic activity, decline of activity of some inhibitors of enzymes, and so on. Some important proteins involved in the pathological process of breast cancer with changed expression may be useful biomarkers, such as alpha-1-antitrypsin, EF- 1-beta, cathepsin D, TCTP, SMT3A, RPS12, and PSMA1, among which SMT3A, RPS12, and PSMA1 were first reported for breast cancer in this study. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption / ionization tandem time-of-flight mass spectrometry (MALDI-TOF / TOF-MS), incorporated with online database searching, were per- formed to investigate differential proteins of breast cancer and adjacent normal breast tissues. Considering that serum albumin is abundantly presented in norma control samples, 15 differential spots detected in 11 out of 12 (91.7%) breast cancer samples were identified by online SIENA-2 DPAGE database searching and The results indicate that pathological changes of breast cancer are concerned with augmentation of substance metabolism, promo- tion of proteolytic activity, decline of activity of some inhibitors of enzymes, and so on. Some important proteins involved in the pathological process of breast cancer with changed expression may be useful biomarkers, such as alpha-1-antitrypsin, EF-1-beta, cathepsin D, TCTP, SMT3A, RPS12, and PS MA1, among which SMT3A, RPS12, and PSMA1 were first reported for breast cancer in this study.