We report that reliable quantitative proteome analyses can be performed with tissue samples stored at -80 degrees C for up to 10 years. However, storing protein extracts at 4 degrees C for 24 h and freezing protein extracts at -80 degrees C and thawing them significantly altered 41.6 and 17.5% of all spot intensities on 2-DE gels, respectively. Fortunately, these storing effects did not impair the reliability of quantifying 2-DE experiments. Nonetheless, the results show that freezing and storage conditions should be carefully controlled in proteomic experiments.

The reliability of 2-DE gel-based comparative proteomics is severely impaired by the potential presence of overlapping proteins. We describe a methodological procedure which may solve this problem. Corresponding protein spots from two experimental groups are digested in the presence of 16O and 18O, respectively. Samples are pooled and proteins identified by MS. The 18O/16O-ratios of the different proteins found in the same spot distinguish proteins with altered from those whose intensity is unchanged.

We performed quantitative comparisons with the two-dimensional gel electrophoresis technique and evaluated the reliability of biostatistical tests for the correction of "false significant" results (alpha-error) by performing repeated runs of an experiment. Results based on uncorrected p-values yielded numerous significant differences in spot intensity which could not be replicated in two additional runs. The best strategy for avoiding these "false-positive" results was strongly dependent on the type of result. In experiments yielding very marked group differences in spot intensity, calculation of the "False Discovery Rate" (FDR) by the Benjamini and Hochberg method corrected the results with sufficient reliability. In experiments yielding relatively small (p-values>0.001) group differences, up to 100% of all results which were significant in two repeated runs were excluded ("false-negative") by calculation of the FDR. In such experiments, significant differences need confirmation by repeated runs.