The number of proteomics studies concerning human brain samples has been increasing in recent years, in particular in the discovery of biomarkers for neurological diseases. The human brain samples are obtained from brain banks, which are interested in providing high quality human nervous tissue. In order to provide brain banks as well as scientists working in the proteomics field with measures for tissue quality, the critical factors after death, the effect of post-mortem interval (PMI) and storage temperature on the human brain proteome were investigated. This study was focused on the gray matter of the frontal cortex. The PMI was artificially prolonged from the time of autopsy (2 h after death) by storing samples at 4 degrees C or room temperature over 18, 24, and 48 h. The samples were analyzed by 2-D DIGE using a pH 4-7 gradient, revealing a time course of quantitative protein changes. The degradation of three proteins, peroxiredoxin-1, stathmin, and glial fibrillary acidic protein were further confirmed by Western-blot analysis. Proteins vulnerable to PMI were analyzed by the 2-D DIGE analysis of cortex samples from three donors, and were derived from a variety of functional groups, including metabolic, structural, stress response, antioxidants, synaptosomal, and neuronal proteins.