Biosynthesis, purification and analysis of selenomethionyl calmodulin by gel electrophoresis-laser ablation-ICP-MS and capillary HPLC-ICP-MS peptide mapping following in-gel tryptic digestion

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Abstract
A biosynthesis method was developed to produce a standard of a selenium-containing protein. It consisted of the expression of calmodulin in Escherichia coli culture in the presence of selenomethionine, which allowed the replacement of all methionine residues by selenomethionine. The resulting 17 kDa protein containing 8 selenomethionine residues was purified by two-step hydrophobic interaction chromatography. The selenomethionyl calmodulin was subsequently used to develop a method for the characterization of selenium-containing proteins (detected in the polyacrylamide gel by laser ablation-ICP-MS) by means of peptide mapping using capillary HPLC-ICP-MS. The monitoring of the 80Se isotope using an ICP mass spectrometer equipped with a collision cell allowed as little as 0.3 pg as Se (1.3 ng ml−1 in the analysed solution) to be detected in the gel. The band containing the protein of interest was excised, the protein was digested with trypsin and the Se-containing peptides were analyzed by capillary HPLC-ICP-MS. The sensitivity of the method was at least a factor of 5 higher than that of capillary LC-electrospray MS/MS in similar conditions. Some of the selenopeptides detected by capillary LC-ICP MS could nevertheless be identified by retention time matching using a set of peptides generated by trypsin digestion from the concentrated selenomethionyl calmodulin standard.