Mass spectrometric identification of amino acid transformations during oxidation of peptides and proteins: modifications of methionine and tyrosine.

Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS), tandem mass spectrometry with on-line liquid chromatography (LC/ESI-MS/MS) and high-resolution mass spectrometry with liquid secondary ionization (LSI-MS) were utilized to identify the modified amino acids in peptides and proteins formed during oxidation with performic acid. The procedure of protein oxidation was chosen to assist in protein unfolding by oxidizing the cystines to cysteic acids to allow for more complete proteolytic digestion and to create additional cleavage sites for endoproteinase Asp-N. Investigation of the Asp-N peptide map of oxidized superoxide dismutase (SOD) by LC/ESI-MS revealed that an expected proteolytic fragment of the protein was missing. In its place, two peptides with molecular weights 66 and 100 higher than that calculated for the missing peptide were observed. To identify the modified amino acids in the unexpected peptides, a model peptide with some amino acid similarities (tyrosine, arginine, methionine, lysine) to the missing peptide was chosen and was subjected to similar oxidation and enzymatic digestion steps, conditions, and reactions. After oxidation and digestion, the model peptide (TAP; sequence, Ac-MDKVLNRY) showed three major peaks in LC/MS. The peptides in the three peaks were identified as the unmodified peptide and two peptides whose molecular weights were 66 and 100 higher than that of TAP.(ABSTRACT TRUNCATED AT 250 WORDS)