LC-MS analysis of the oxidation ratio of methionine-containing peptides

To quantify [Met(O)]/[Met], digested peptides were injected onto a 2.0 mm (inner diameter) × 50 mm CAPCELL PACK MGIII-H S3 column attached to a Nanospace SI-2 HPLC system (Shiseido Fine Chemicals, Tokyo, Japan). The column temperature was maintained at 45 °C. The flow rate of the mobile phase was 200 μL/min; mobile phase A consisted of 0.05% FA and mobile phase B consisted of 0.05% FA/90% ACN. The mobile phase gradient was programmed as follows: 0% B (0–3 min), 0–55.5% B (3–40 min), 55.5–80% B (40–40.1 min), and 80% B (40.1–45 min). Peptides were introduced from the HPLC to an LTQ-Orbitrap Discoverer. Full-scan MS spectra (m/z 300–2,000) were acquired using an Orbitrap instrument at a mass resolution of 30,000 at m/z 400.