ELECTROPHORESIS-RELATED PROTEIN MODIFICATION - ALKYLATION OF CARBOXY RESIDUES REVEALED BY MASS-SPECTROMETRY

In recent years, the combination of gel electrophoresis and mass spect rometry has developed into one of the most powerful approaches for the analysis of proteins. However, a number of gel electrophoresis-induce d protein modifications have been described. Cystein is the most endan gered amino acid readily reacting with mercaptoethanol or free acrylam ide. In the course of studies on glucan phosphorylases (E.C.2.4.1.1) f rom white potato (Solanum tuberosum L.) and the T cell receptor, we no ticed that proteolytic peptides from these proteins can undergo an une xpected modification, giving rise to a mass increment of 14 Da. By pos t-source decay (PSD) analysis the modification was identified as methy lation of the glutamic acid side chain carboxyl group. The methylation takes place during Coomassie blue staining of proteins if both trichl oroacetic acid and methanol are present in the staining solution. Repl acement of methanol by ethanol under otherwise unchanged conditions re sults in ethylation of the peptides. The in vitro alkylation was furth er studied by using synthetic peptides which contain, at different pos itions: glutamic acid, aspartic acid or the corresponding amides. The kinetic analysis of the observed reactions revealed that glutamic acid is preferentially methylated. The three other amino acid residues can be methylated but with a velocity at least one order of magnitude low er. Although these modifications complicate the interpretation of the spectra, they provide valuable structural information.