Nanoviper c18 separation column

The fractions were separated using an EASY-nLC 1000 system (Thermo Fisher Scientific). The binary buffer system included an A phase (0.1% formic acid in 2% acetonitrile) and B phase (0.1% formic acid in 84% acetonitrile). Peptides were re-dissolved in the A phase and loaded into a reversed-phase analytical pre-column (Acclaim PepMap100, 100 μm inner diameter × 2 cm, Nanoviper® C18 separation column, Thermo Fisher Scientific). Next, each fraction was directly injected into and analysed by the Easy Column (10 cm × 79 μm, 3 μm particle size, Thermo Fisher Scientific) at a flow rate of 330 nL/min. The LC-MS/MS analysis was performed using a Thermo Fisher Scientific Q-Exactive high resolution mass spectrometer. The MS scan was set in the positive ion mode (300–1800 m/z). The resolutions of MS1 and MS2 were 70,000 at 200 m/z and 17,500 at 200 m/z, respectively. For MS1, the automatic gain control (AGC) target value, maximum ion injection time and dynamic exclusion were set to 1 × 10⁶, 50 ms and 60 s, respectively. MS data were acquired by full-MS scan followed by 20 MS2 spectra. For MS2, each activation type was HCD and the collision energy was 30 eV for all ions. The isolation window and underfill ratio were defined as 2m/z and 0.1%, respectively.

LC-MS/MS experiments were performed using a Sciex 5600⁺ triple-time-of-flight (TOF) mass spectrometer coupled with a ChromXP reversed-phase 3-μm C₁₈-CL trap column (350 μm by 0.5 mm, 120 Å; Eksigent; AB Sciex, MA, USA) and a nanoViper C₁₈ separation column (75 μm by 250 mm, 3 μm, 100 Å; Acclaim PepMap; Thermo Fisher Scientific, MA, USA) in an Eksigent nanoLC (ultra 2D plus) system. The binary mobile solvent system used was as follows: solvent C, 2% (vol/vol) acetonitrile, 0.1% (vol/vol) formic acid in water; solvent B, 98% (vol/vol) acetonitrile, 0.1% (vol/vol) formic acid. The peptides were separated at a flow rate of 200 nl/min in a 60-min gradient with a total run time of 90 min. The MS data of each condition were acquired in IDA (information-dependent acquisition) with high sensitivity mode. Each cycle consisted of 250- and 100-ms acquisition times for MS1 (m/z 350 to 1,250 Da) and MS/MS (100 to 1,800 m/z) scans, respectively, with a total cycle time of ∼2.3 s. Each condition was run in triplicate.