Proteolysis is one of the major problems in collection and storage of biological samples for proteome analysis, particularly when the samples undergo freeze-thaw cycles. The use of protease inhibitors for prevention of such proteolysis in some samples is debated because protease inhibitors may interfere with proteome analysis and whether protease inhibitors are useful for renal and urinary proteomics remains unclear. We therefore performed a systematic evaluation of the use of protease inhibitors in gel-based renal and urinary proteomics. Renal proteins were extracted from porcine kidney tissue and stored at -30 or -70 degrees C without protease inhibitors. After 0, 2, 4, 6, 8, 10, and 12 freeze-thaw cycles, the 2-D proteome profile was examined. Differential spot analysis and ANOVA with Tukey posthoc multiple comparisons revealed significantly quantitative changes in intensity levels of 12 and 7 renal proteins that were stored at -30 and -70 degrees C, respectively, after >or=4 freeze-thaw cycles. Additionally, there were qualitative changes (vertical elongation or streak) in 6 and 1 renal proteins that were stored at -30 and -70 degrees C, respectively. All these changes could be successfully prevented by the addition of 1% (v/v) protease inhibitors cocktail prior to storage. In contrast, neither quantitative nor qualitative changes were observed in urine samples that were stored without protease inhibitors and processed as for kidney samples. From these data, the addition of protease inhibitors is highly recommended for gel-based renal proteomics, but no longer recommended for gel-based urinary proteomics.