Q exactive plus system

The tryptic peptides were fractionated by high pH reverse-phase HPLC using an Agilent 300 Extend C18 column (5 μm particles, 4.6 mm ID, and 250 mm length). The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a custom-made reversed-phase analytical column. The gradient involved an increase from 7% to 25% solvent B (0.1% formic acid in 98% acetonitrile) for 38 min and from 25% to 40% for 14 min, after which it increased to 80% for 4 min and then was maintained at 80% for the last 4 min, all at a constant flow rate of 700 nL/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) by using a Q Exactive^TM Plus system (Thermo, Waltham, MA, USA) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1000 for the full scan, and the intact peptides were detected by the orbitrap instrument at a resolution of 70,000. The peptides were then selected for MS/MS using the NCE setting at 27, and the fragments were detected by the orbitrap instrument at a resolution of 17,500. The maximum IT was set at 20 s for the full MS and to “auto” for MS/MS. The isolation window for MS/MS was set at 2.0 m/z.

Peptides were dissolved in 0.1% formic acid (solvent A) and loaded directly onto a homemade reversed-phase analytical column (15-cm length, 75 μm inner diameter). Samples were then eluted at 350 nL/min using a mobile phase consisting of 0.1% formic acid in 98% acetonitrile solvent B under the control of an EASY-nLC 1000 UPLC system (Thermo Fisher Scientific). The elution protocol was as follows: 9–26% solvent B for 40 min, 26–35% solvent B for 14 min, 35–80% solvent B for 3 min, and holding at 80% for the last 3 min. Eluted peptides were then subjected to nanoelectrospray ionization (NSI) followed by tandem mass spectrometry (MS/MS) using the Q ExactiveTM Plus system (Thermo Fisher Scientific) coupled to the UPLC. The electrostatic voltage applied was 2.1 kV and the m/z scan range was 400 to 1500. Both intact peptides and fragments were detected in the Orbitrap at resolutions of 70,000 and 35,000 FWHM, respectively. Peptides were then selected for MS/MS using a normalized collision energy (NCE) setting of 28. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 1 × 10⁴ in the MS survey scan with 30.0 s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed first mass was set as 100 m/z.

The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a self-made reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient consisted of 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) in 26 min, 23% to 35% in 8 min, and to 80% in 3 min. For the last 3 min, 80% solvent B was used at the constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system.
The peptides were subjected to an NSI source followed by MS/MS in a Q Exactive Plus system (Thermo Fisher Scientific) coupled online to the UPLC. The electrospray voltage was 2 kV. The m/z scan range was 350 to 1,800 for the full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using an NCE setting of 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure was used that alternated between one MS scan followed by 20 MS/MS scans with 15-s dynamic exclusion. The automatic gain control (AGC) was set at 5E4 and the fixed first mass was set as 100 m/z.