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.