Conversion of methionine into homocysteic acid in heavily oxidized proteomics samples

Abstract

Analysis of protein oxidation is necessary in numerous areas of biochemistry, including hydroxyl radical surface mapping, oxidative stress assays, and pharmaceutical stability testing. Mass spectrometry is one of the tools most often used to identify protein oxidation products, and previous studies have attempted to identify and characterize all of the major oxidation products detected by mass spectrometry for each amino acid residue. In this note, we present evidence that in heavily oxidized protein samples, such as those produced by hydroxyl radical surface mapping, a major oxidation product of methionine is homocysteic acid. The formation of homocysteic acid from methionine was previously unrecognized in other mass spectrometric analyses, and has important implications for the analysis of oxidized samples, as well as potential implications as to the functional consequences of methionine oxidation.

Figure 1

(a) MS/MS spectrum (Thermo LTQ Orbitrap) from a Cyp3A4 sample from microsomes, prepared by gel electrophoresis. The precursor mass (M+H), measured by the Orbitrap, is 1169.579 Da. M[+33.969] gives a theoretical precursor mass of 1169.577 Da. Peaks for the intense fragment ions (b2, y2, b3, b4, y4, etc.), measured by the LTQ analyzer, are all within 0.13 Da of their theoretical values. The peptide is a tryptic peptide from cytochrome P450. (b) MS/MS spectrum (Thermo LTQ) from a hydroxyl radical surface mapping experiment on horse myoglobin. Peaks for the intense singly charged ions (b2, y2, b4, y3, etc.) are all within 0.14 Da of their theoretical values. We interpret the peak at 727.5 labeled "M+2H-82" as the precursor with a neutral loss of sulfonic acid.

Figure 2

(a) Positive ion mode mass spectra of 30μM oxidized Gly-Met-Gly (GMG). (b) Negative ion mode mass spectra of 30μM oxidized GMG.

Figure 3

Proposed scheme for radiolytic oxidation of methionine to homocysteic acid.