Identification of the modified amino acid residue in the modified heme protein using LC/MS/MS

An amino acid in a heme protein can be modified by reaction with hydrogen peroxide, resulting in loss of physiological activity of the protein. Additionally, it has been reported that the insertion of tryptophan near the heme in myoglobin can be oxidized by an organic acid peroxide.
In this study, we found that a methionine residue is modified in a mutant of another heme protein. However, among the peptide fragments obtained by the peptidase digestion of the modified protein, two peptides were present with the same molecular weight, containing methionine and constructed with the same set of amino acids. From this we identified the modified amino acid in the mutant protein using LC-MS/MS.
The mutant of the heme protein used in this study was a mixture of modified and non-modified proteins, with about 50% of the native protein oxidized. After digesting the mutant of the heme protein with trypsin, the obtained peptide fragments were analysed by mass spectrometry. The sample was analyzed using a double-focusing mass spectrometer with electrospray ionization. Then LC-MS/MS analysis was performed using UHPLC (Nexera, Shimadzu Corporation, Japan) and a triple quadruple mass spectrometer (LCMS-8030, Shimadzu Corporation, Japan). Separation was achieved using an ODS column, Shim-pack XR-ODS II (150 x 2.0 mm, 2.2um). The sample was eluted at 0.2 mL/min with a binary gradient system and analysed by MS/MS in positive ion electrospray.
The tryptic digested sample of the heme protein was analyzed by ESI-MS. Peptide F2 (sequence: IFIMK) and peptide F8 (sequence: MIFIK) at m/z 651 (M+H) differed in sequence. Peak at m/z 667, which was 16u larger than the original peak is likely to be due to the oxidized species of peptide F2 or F8. MS/MS analysis of two products at m/z 326 (M+2H) corresponded to the mass of the peptides F2 or F8, and the peak at m/z 334 (M+2H) to the modified peptide. In order to analyze the sequence of the peptide exhibiting these peaks, product ion scan (MS/MS) measurements were performed with three collision energies (CE=10, 20, 30V) and a high scan rate (15000u/sec). The results of product ion scan data showed that the two peaks at m/z 326 were F2 and F8, respectively, and that the peak of m/z 334 was F2 (IFIMK).