MASS-SPECTROMETRIC IDENTIFICATION OF AMINO-ACID TRANSFORMATIONS DURING OXIDATION OF PEPTIDES AND PROTEINS - MODIFICATIONS OF METHIONINE ANDTYROSINE

Liquid chromatography/electrospray ionization mass spectrometry (LC/ES I-MS), tandem mass spectrometry with on-line liquid chromatography (LC /ESI-MS/MS) and high-resolution mass spectrometry with liquid secondar y ionization (LSI-MS) were utilized to identify the modified amino aci ds in peptides and proteins formed during oxidation with performic aci d. 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 site s for endoproteinase Asp-N. investigation of the Asp-N peptide map of oxidized superoxide dismutase (SOD) by LC/ESI-MS revealed that an expe cted proteolytic fragment of the protein was missing. In its place, tw o peptides with molecular weights 66 and 100 higher than that calculat ed for the missing peptide were observed. To identify the modified ami no acids in the unexpected peptides, a model peptide with some amino a cid similarities (tyrosine, arginine, methionine, lysine) to the missi ng peptide was chosen and was subjected to similar oxidation and enzym atic digestion steps, conditions, and reactions. After oxidation and d igestion, 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 wer e 66 and 100 higher than that of TAP. The LC/ESI-MS/MS of these reacti on products demonstrated that in the two modified peptides the N-termi nal methionine has been transformed into methionine sulfone (molecular weight increase of 32), whereas m/z values of ah the fragment ions co ntaining C-terminal arginine and tyrosine were 34 and 68 higher than t hose in the unmodified peptide. To establish whether the arginine or t yrosine or both were modified, another peptide neuromedin N (sequence, KIPYIL) was chosen that contains tyrosine but no arginine or methioni ne. The LC/MS analysis of the oxidized peptide again showed three peak s. The early-eluting peak corresponds to residual unmodified peptide a nd the molecular weight of the two later-eluting peptides are respecti vely 34 and 68 higher than that of neuromedin N. The LC/ESI-MS/MS of t he peptides confirmed that the tyrosine underwent transformations with an increase of molecular weight by 34 or 68. An accurate molecular we ight measurement together with the determination of the atomic composi tion of the modified peptides showed the presence of one and two chlor o substitutions in tyrosine in the two later-eluting peptide peaks, re spectively. 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 chlor ide used as buffer in peptides and SOD solutions produced electrophile s in the oxidizing medium that caused electrophilic aromatic substitut ion in tyrosine. This observation was further supported by the identif ication of mono and dibromo peptides when chloride salts were replaced with bromides.