| Abstract: | An electrochemical flow cell coupled on-line to a mass spectrometer is used to oxidize a range of proteins. Oxidation of tyrosine and tryptophan can give rise to peptide bond cleavage at their C-terminal side. This suggests the possible use of electrochemistry as an alternative protein digestion method. For the small proteins insulin and alpha-lactalbumin (6 and 14 kD) almost all potential sites are cleaved, while for the largest successfully tested protein (carbonic anhydrase, 29 kD) 7 of the 15 available sites were specifically cleaved. Several proteins did not produce peptides upon electrochemical oxidation, possibly due to problems with accessibility of tyrosine and tryptophan residues, or to competing oxidation reactions. Peptides were generally not the major oxidation products: non-cleavage oxidation products observed as protein mass + n x 16 Da, presumably by oxidation of tyrosine, tryptophan, cysteine and methionine, account for the major fraction of protein oxidation products. Nevertheless the amount and variety of cleavage products at the present conditions shows good prospects for further improvement of the system. The efficient protein oxidation also allows the use of the EC-MS system as a tool to study protein oxidation reactions in general. The preconditioning and life history and/or age of the electrochemical cell was relevant to the solvent and sample conditions needed for efficient oxidative cleavage as opposed to other oxidation reactions. |
