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The peptides were loaded onto a reverse phase trap column (Thermo Scientific Acclaim PepMap100, 100 m*2 cm, nanoViper C18) connected to the C18-reversed-phase analytical column (Thermo Scientific Easy Column) in buffer A (0.1% Formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nl/min controlled by IntelliFlow technology