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Running time was 22 min, flow rate was 1.5 ml.min-1.
Exposure to the smoke lasted 45 min (flow rate of 100 mL·min−100
The column was developed with a gradient of acetonitrile in 5 mM TPA pH 7.0 (0–10% in 20 min, flow rate 1 mL/min).
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Peptides were eluted using a 5 to 50% gradient of solvent B during 80 min at 300 nl/min flow rate.
The gradient consisted of 0% B to 80% B in 0 40 min at 4.0 mL/min flow rate.
After loading the sample, the column was washed with 90% solvent B for 15 min at 0.5 mL/min flow rate.
It was applied using a linear 11 min gradient from 90% solvent A to 30% solvent A (1 to 12 min) at a 0.35 mL/min flow rate.
Peptides were eluted using a 4-50% gradient of solvent B during 30 min at a 300 nl/min flow rate.
After loading the sample, the column was washed with 98% solvent A for 10 min at a 1 mL/min flow rate.
Then, the perfusion solution I without EGTA was supplemented with 5 mM CaCl2.2H2O and 0.01% collagenase type 4 (Worthington Biochemical Corporation, Serlabo Technologies, Entraigues, France) at a 7 mL/min flow rate for 5 7 min.
The mobile phase was methanol/water (90 10, v/v) at 1.0 ml/min flow rate for 30 min.
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