A reactive, surface cysteine residue of the class-II fructose-1,6-bisphosphate aldolase of Escherichia coli revealed by electrospray ionisation mass spectrometry.

The state of post-translational modification of the class-II fructose-1,6-bisphosphate aldolase (FBP-aldolase) purified from Escherichia coli was examined by electrospray ionisation mass spectrometry (ESI-MS). The mass was larger than that expected from the known DNA sequence by approximately 80 +/- 6 Da, suggesting the presence of a covalent modification on the protein. Phosphorylation (+ 80 Da), a known modification in an FBP-aldolase from Bacillus subtilis and a suspected modification in this E. coli aldolase, was ruled out as the extra mass was readily removed by treatment with dithiothreitol. Purification of aldolase by a protocol which omitted 2-mercaptoethanol from all buffers resulted in the purified protein having the expected mass (39016 Da). The extra mass was therefore established as a covalent adduct of the protein with 2-mercaptoethanol (+ 76 Da). Reduction and alkylation studies, followed by isolation of tryptic peptides, established that the site of attachment was Cys36. Although no significant effect of the modification on the activity of the protein was observed, the study underlines the ease with which a protein can be modified covalently by a simple and mild purification procedure; such labelling, which may not always be benign, would be undetectable without the routine use of mass spectrometric analysis.