Proteome analysis differentiates between two highly homologues germin-like proteins in Arabidopsis thaliana ecotypes Col-0 and Ws-2

A proteome approach based on 2-D gel electrophoresis (2-DE) was used to compare the protein patterns of the Arabidopsis ecotypes Col-0 and Ws-2. In leaf extracts a pair of protein spots were found to be diagnostic for each of the lines. Both pairs of spots were identified as closely related germin-like proteins differing in only one amino acid by using peptide mass finger printing of tryptic digests and by gaining additional data from post-source decay spectra in the MALDI-TOF analysis. Western blot analysis after separation of protein extracts by 2-DE confirmed results from Coomassie blue-stained gels and revealed additional immunoreactive spots for both ecotypes most likely representing dimers of the spots first identified. Western blot analysis and mass spectrometrical identification of the corresponding weakly stained protein in Coomassie blue-stained gels of the ecotype Col-0 also demonstrated for the first time the occurrence of AtGER3 protein in root extracts. Our results demonstrate the capacity of proteome analysis to analyse and distinguish closely related members of large protein families.