Eksigent nanolc 1d plus system

All the analyses were performed using a Triple TOF 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA) equipped with a NanoSpray III source (AB SCIEX, Framingham, MA, USA). The samples were loaded onto a capillary C18 trap column (3 cm × 100 µm) and then separated by a C18 column (15 cm × 75 µm) on an Eksigent nanoLC-1D plus system (AB SCIEX, Framingham, MA, USA). The flow rate was 300 nL/min, and the linear gradient was set as follows: 0–0.5 min, 95–92% A; 0.5–48 min, 92–74% A; 48–61 min, 74–62% A; 61–61.1 min, 62–15% A; 61.1–67 min, 15% A; 67–67.1, 15–95% A; and 67.1–70 min, 95% A. Mobile phases A and B consisted of 2% Acetonitrile (ACN)/0.1% Formic acid (FA) and 95% ACN/0.1% FA, respectively.
The data were acquired with an ion spray voltage of 2.4 kV, a curtain gas pressure of 35 psi, a nebulizer gas pressure of 5 psi, and an interface heater temperature of 150 °C. The MS scan was performed between 400 and 1,500 with an accumulation time of 250 ms. For IDA, 30 MS/MS spectra (80 ms each, mass range of 100–1,500) of MS peaks with an intensity higher than 260 and a charge state between 2 and 5 were acquired. A rolling collision energy voltage was used for CID fragmentation to acquire the MS/MS spectra. The mass was dynamically excluded for 22 s.

Peptides were separated on an Agilent 1200 HPLC with an Agilent column (Wilmington, DE, United States). All analyses were performed on a Triple TOF 5600 System (AB Sciex) fitted with a NanoSpray III source (AB Sciex). Samples were loaded by a capillary C18 trap column and then separated by a C18 column on an Eksigent NanoLC-1D plus system (AB Sciex) using an autosampler with a flow rate of 300 nl/min. Ion spray voltage was 2.5 kV, MS scans were acquired in 250 ms, and product ion scans that exceeded a threshold of 150 counts per second with a two to five charge state were collected at most 35 times. Cycle time was fixed to 2.5 s. Specific parameters were as previously described (Li et al., 2018 ).

LC–MS/MS analysis was performed on a TripleTOF 5,600 mass spectrometer (SCIEX, USA) coupled with a Nano spray III source (SCIEX, USA) for 60 min. A capillary C18 trap column (3 cm × 100 µm) following with a C18 column (15 cm × 75 µm) on an E ksigent nanoLC-1D plus system (SCIEX, USA) was utilized for separation. The mobile phase was made of solvent A (0.1% formic acid in water) and solvent B (ACN-H2O-FA, 80: 19.9: 0.1, v/v/v) and remained a constant flow rate at 300 nL/min. Linear gradient was adjusted as follows: 0–45 min, 5–26% B; 45–52 min, 26–60% B; 52–53 min, 60–95% B; 53–60 min, 95% B.
The mass spectrometer was performed both in positive ion mode, and parameters were established as follows: spray voltage: 2.5 kV ( +); automatic gain control (AGC) target: 2 × 10⁵ ions; centroid mass data with full scan: m/z 350–1950; MS/MS mode collision energy: 38 eV; peptide detection resolution of MS/MS: 15,000; automatic gain control (AGC) target: 5 × 10⁴ ions; maximum ion injection time (IT): 100 ms; dynamical exclusion time for Mass was set at 60 s.