The present study was designed to reveal protein modifications in control cases related with postmortem delay and temperature of storage in 3 paradigms in which the same postmortem tissue sample (frontal cortex) was frozen a short time after death or stored at 1 degrees C, 4 degrees C, or room temperature and then frozen at -80 degrees C at different intervals. No evidence of protein degradation as revealed with monodimensional gel electrophoresis and Western blotting was observed in samples artificially stored at 1 degrees C and then frozen at different intervals up to 50 hours after death. However, the levels of several proteins were modified in samples stored at 4 degrees C and this effect was more marked in samples stored at room temperature. Two-dimensional gel electrophoresis and mass spectrometry further corroborated these observations and permitted the identification of other proteins vulnerable or resistant to postmortem delay. Finally, gel electrophoresis and Western blotting of sarkosyl-insoluble fractions in Alzheimer disease showed reduced intensity of phospho-tau-specific bands with postmortem delay with the effects being more dramatic when the brain samples were stored at room temperature for long periods. These results emphasize the necessity of reducing the body temperature after death to minimize protein degradation.