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Briefly, the peptide samples were trapped and washed on Pepmap100 C 18 trap (5m, 0.3 5 mm) at 20ul/min using 2% acetonitrile in water (with 0.1% formic acid) for 10 min and then separated on a Pepman100 RSLC C18 column (2.0 m, 75-m 150-mm) using a gradient of 2%40% acetonitrile with 0.1% formic acid over 120 min at a flow rate of 300 nL/min and a column temperature of 40C

Therefore, SetCys peptide 2 is constantly present in solution and escapes the SetCys/SetCys amide equilibrium by irreversibly losing its N -selenoethyl limb as discussed above