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Peptides were eluted over a 120-min gradient with buffer A as 100% water + 0.1% formic acid and buffer B as water + 0.1% formic acid and acetonitrile with the following steps: 0–5% B for 1 min, 5–25% for 68 min, 25–40% for 20 min, 40–60% for 15 min, 60–95% for 1 min and 95% for 15 min