Electrospray mass and tandem mass spectrometry identification of ozone oxidation products of amino acids and small peptides

Abstract

Aqueous ozonation of the 22 most common amino acids and some small peptides were studied by electrospray mass (ESI-MS) and tandem mass spectrometry. After 5 min of ozonation only His, Met, Trp, and Tyr form oxidation products clearly detectable by ESI-MS. For His, the main oxidation product is formed by the addition of three oxygen atoms, His + 3O; for Met and Tyr by the addition of one oxygen atom, Met + O and Tyr + O, and for Trp by the addition of two oxygen atoms, Trp + 2O. Ozone oxidation occurs rapidly, products are already detected after 30 s of ozonation, and the reactivity order is Met > Trp > Tyr > His. The structures of the oxygen addition products were investigated by electrospray product ion mass spectra, and by comparing these spectra to those of protonated intact amino acids, and when available, to those of model compounds. His + 3O was assigned as 2-amino-4-oxo-4-(3-formylureido)butanoic acid ( 1) formed by oxidation of the His imidazole ring, Met + O as methionine sulfoxide ( 2), Trp + 2O as N-formylkynurenine ( 4), and Tyr + O as a mixture of dihydroxyphenylalanines ( 7 and 8). Ozonation of peptides show that the same number of oxygen atoms are added as expected from the ozonation of the free amino acids. The product ion mass spectra of both the protonated intact peptides, MH +, and the main ozonation products (M + nO)H + ( n = 1–3) revealed b and y type ions as the main fragments, which allow one to assign the type and location of modified amino acid in the model peptides.