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. The LC/ESI-MS/MS of these reaction products demonstrated that in the two modified peptides the N-terminal methionine has been transformed into methionine sulfone (molecular weight increase of 32), whereas m/z values of ah the fragment ions containing C-terminal arginine and tyrosine were 34 and 68 higher than those in the unmodified peptide. To establish whether the arginine or tyrosine or both were modified, another peptide neuromedin N (sequence, KIPYIL) was chosen that contains tyrosine but no arginine or methionine. The LC/MS analysis of the oxidized peptide again showed three peaks. The early-eluting peak corresponds to residual unmodified peptide and the molecular weight of the two later-eluting peptides are respectively 34 and 68 higher than that of neuromedin N. The LC/ESI-MS/MS of the peptides confirmed that the tyrosine underwent transformations with an increase of molecular weight by 34 or 68. An accurate molecular weight measurement together with the determination of the atomic composition of the modified peptides showed the presence of one and two chloro substitutions in tyrosine in the two later-eluting peptide peaks, respectively. This finding is in agreement with the detection of 3- and 3,5-dichlorotyrosines in acid hydrolysis (Sanger, F.; Thompson, E. O. P. Biochim. Biophys. Acta 1963, 71, 468). We believe that sodium chloride used as buffer in peptides and SOD solutions produced electrophiles in the oxidizing medium that caused electrophilic aromatic substitution in tyrosine. This observation was further supported by the identification of mono and dibromo peptides when chloride salts were replaced with bromides.