Easy nanolc 1200 instrument

The liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were performed on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled with an online EASY-nanoLC™ 1200 instrument (Thermo Fisher Scientific, Waltham, MA, USA). The method of parameter configuration was the same as that previously described [33 (link)]. Peptides were separated on a nanoViper PepMap100 C18 column (75 µm × 25 cm, Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase consisted of 0.1% formic acid (Thermo Fisher Scientific, Waltham, MA, USA) in water (A) and 0.1% formic acid/80% acetonitrile (B) (Thermo Fisher Scientific, Waltham, MA, USA). The gradient profile was set as follows: 3–7% B for 2 min, 7–35% B for 83 min, 35–68% B for 20 min, 68–100% B for 10 min and equilibrated in 100% B for 15 min. Mass Spectrometry (MS) analysis was performed using an Orbitrap Fusion Lumos mass spectrometer. The spray voltage was set at 2.3 kV. Orbitrap MS1 spectra (Automatic Gain Control 4 × 10⁵) were collected from 350–1800 m/z at a resolution of 60 K followed by data-dependent HCD MS/MS (resolution 15,000, collision energy 30%) using an isolation width of 1.6 m/z. A dynamic exclusion time was set to 45 s.

Each peptide sample underwent triplicate LC−MS/MS runs using an Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific, Germany) coupled with an online EASY-nanoLC™ 1200 instrument (Thermo Fisher Scientific, Germany). Samples were first loaded onto a 75 μm × 2 cm nanoViper PepMap™100 C18 precolumn and then separated on a 75 μm × 50 cm nanoViper PepMap™100 C18 analytical column (Thermo Fisher Scientific, Germany). Mobile phase consisted of 0.1% FA (A) and 0.1% FA/80% ACN (B). The gradient profile (240 min) was set as follows: 3–7% B for 2 min, 7–35% B for 166 min, 35–68% B for 40 min, 68–99% B for 10 min, and 99% B for 22 min. The parameters of mass spectrometry were set as follows: for MS1, scan range of orbitrap spectra (automatic gain control AGC 4 × 10⁵) were from 350 to 1,800 m/z at a resolution of 60 K. For MS2, the multiply charged ions were fragmented in the collision cell by higher-energy collisional dissociation (HCD, collision energy 30%) with an isolation window of 1.6 m/z, a maximum injection time of 30 ms, a resolution of 15 K and AGC target of 5 × 10⁴.