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Peptides were separated by loading into the trap column in a loading buffer for 5 min at a 5 L/min flow rate and eluted from the analytical column at a flow rate of 150 L/min using a 130 min LC gradient as follows: linear gradient of 5% to 27% of solvent B in 80 min, 2745% in next 20 min, 45100% of B in next 5 min at which point the gradient was held at 100% of B for 7 min before reverting back to 2% of B at 112 min, and held at 2% of B for next 18 min for equilibration