Quantitative proteome analysis of Alzheimer's disease (AD) brains was performed using two-dimensional (2-D) gels in order to find out the pathological protein expression in AD. We sequentially extracted brain proteins using two distinct sample solutions, yielding different protein fractions (fraction A and B). These fractions showed distinct 2-DE patterns with high resolution and excellent reproducibility. In fraction A (solubilized by urea and Nonidet P-40 (NP-40)), approximately 1300 protein spots were detected, and the relative volume (%VOL) significantly increased in five spots and significantly decreased in 10 spots in AD. The proteins identified include enzymes, molecular chaperones and cytoskeletal proteins. In fraction B (solubilized by urea, thiourea, N-decyl-N,N-dimethyl-3-ammonio-1-propane sulfonate (SB3-10) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)), over 500 protein spots were detected in the 2-DE data analysis. The %VOL of three spots was significantly increased in AD. Two of these spots were identified as glial fibrillary acidic protein (GFAP) using mass spectrometry. These results suggest that subproteomics following sequentially extracted brain proteins is a useful method for the analysis of brain extracts containing hydrophobic proteins. Our findings will prompt further study on disease-linked proteins for the investigation of AD pathogenesis and the quest for disease markers.