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.

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To understand molecular immune response of Penaeus vannamei during Taura syndrome virus (TSV) infection, expression and functional proteomics studies were performed on hemocyanin, which is a major abundant protein in shrimp hemocytes. Two-dimensional electrophoresis (2-DE) revealed up-regulation of several C-terminal fragments of hemocyanin, whereas the N-terminal fragments were down-regulated during TSV infection. 2-D Western blot analysis showed that the C-terminal hemocyanin fragments had more acidic isoelectric points (pI), whereas the N-terminal fragments had less acidic pI. Further analysis by NetPhos showed a greater number of serine phosphorylation sites in the C-terminal hemocyanin. Additionally, motif scan using Scansite revealed ERK D-domain, which is required for activation of ERK1/2 effector kinase, as a kinase-binding site at the 527th valine in the C-terminal hemocyanin, whereas neither motif nor functional domain was found in the N-terminus. Co-immunoprecipitation confirmed the interaction between the C-terminal hemocyanin and ERK1/2. 1-D Western blot analysis showed that ERK1/2 was also up-regulated during TSV infection. Our findings demonstrate for the first time that ERK1/2 signaling pathway may play an important role in molecular immune response of P. vannamei upon TSV infection through its interaction with the C-terminal hemocyanin.