The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reversed-phase analytical column (15 cm length, 75 μm internal diameter). The gradient was comprised of an increasing from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min, climbing to 80% in 3 min, and then holding at 80% for the last 3 min. The experiment was processed at a constant flow rate of 400 nL/min on an EASY-nLC 1,000 UPLC system (Thermo, Shanghai, China).
The peptides were subjected to NSI source followed by MS/MS in Q ExactiveTM Plus (Thermo, Shanghai, China) coupled online to UPLC. The electrospray voltage applied was set at 2.0 kV. The m/z scan range was set from 350 to 1,800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control was set at 5E4. Fixed first mass was set as 100 m/z31 (link). The MS proteomics data have been set to the ProteomeXchange Consortium by PRIDE partner repository program under identifier PXD016423.
The peptides were subjected to NSI source followed by MS/MS in Q ExactiveTM Plus (Thermo, Shanghai, China) coupled online to UPLC. The electrospray voltage applied was set at 2.0 kV. The m/z scan range was set from 350 to 1,800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control was set at 5E4. Fixed first mass was set as 100 m/z31 (link). The MS proteomics data have been set to the ProteomeXchange Consortium by PRIDE partner repository program under identifier PXD016423.
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