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The column was then washed with buffer A (300 L, 2,000 g , 2 min), and 10 mM aqueous NH 4 HCO 3 containing 5% CH 3 CN (300 L, 2000 g , 2 min), before peptides were eluted from the column as 15 fractions using 300 L buffer containing an increasing concentration of CH 3 CN in 10 mM NH 4 HCO 3 (%CH 3 CN = 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 35, 40, 45, 50, 75) (2000 g , 2 min)