A 60-minute gradient was employed with a linear gradient starting at 30 minutes and consisting of mobile phase A and mobile phase B (0.26 M FA, 10% ACN, 1 M ammonium formate; pH-4-5) for elution of peptides (flow rate 200 uL/min).
A 90-minute linear gradient was then applied from 5 to 40% acetonitrile in 0.1% formic acid at 300 nL/min.
This condition was held for 5 minutes, and the gradient was returned to 100% buffer A in 5 minutes.
For the first 2 minutes, isocratic elution was carried out using 100% of A. From 2 to 5 minutes, a linear gradient was applied using 100 to 30% A in B. From 5 to 5.5 minutes, a linear gradient was applied using 30 to 0% A in B. In the final minute concentration of A was returned to 100%.
For the first two minutes, isocratic elution was carried out using 10% A in B. From 2 to 25 minutes, a linear gradient was applied using 10 to 30% A in B. From 25 to 27 minutes, a linear gradient was applied using 30 to 70% A in B. The volume of the sample injected was 20 μl.
Briefly, a 60-minute, three-step gradient was used to load peptides onto the column via an EASY-nLC pump (Proxeon Biosystems, FL), and peptides were analyzed by a multiplexed SRM method using the following parameters: predicted CE values, 0.002 m/z scan width, 0.01 s scan time, 0.3 Q1, 0.7 Q3, 1.5 mTorr Q2 pressure and tuned tube lens values.
20 mM KH2PO4 (pH 2) was used as the eluent at a flow of 1.5 ml/min, with a 0 - 20% acetonitrile gradient developing between 0 - 6 min, followed by 20% acetonitrile for a further minute, after which the gradient was decreased to 0% within the final minute.
The initial mobile phase composition was 100% A for 3.5 minutes then a linear gradient was applied for 5.5 minutes to 90% B. This was maintained for 1.0 minute and a quick linear gradient back to 100% A for 0.5 minute was followed by a 3.5 minutes equilibrium period.
The gradient was centrifuged for 90 minutes at 35000 rpm.
The gradient was centrifuged for 30 minutes at 1100×g at room temperature with the brake turned off.
