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Subsequent peptide separation was performed on the ACQUITY BEH C18 (2.1 50 mm) analytical column using a first gradient ranging from 9 to 45% of buffer B (80% acetonitrile and 0.1% formic acid) for 10 min followed by a second gradient ranging from 45 to 99% of buffer B for 1 min, at an overall flow rate of 50 L/min