Nanolc 1d plus system

All analyses were performed by a Triple TOF 5600 mass spectrometer equipped with a Nanospray III source. Samples were separated by a reverse-phase C18 column (15 cm × 75 μm, 3 μm, 120 Å) on an Eksigent nanoLC-1D plus system. Mobile phase A = 2% ACN/0.1% FA and B = 95% ACN/0.1% FA. The flow rate was 300 nL/min and linear gradient was set as described previously (Hu et al., 2018 (link)).
Data were acquired with a 2.4 kV ion spray voltage, 35 psi curtain gas, 5 psi nebulizer gas, and an interface heater temperature of 150°C. The MS scanned between 400 and 1500 with an accumulation time of 250 ms. For IDA, 30 MS/MS spectra (80 ms each, mass range 100–1500) of MS peaks above intensity 260 and having a charge state of between 2 and 5 were acquired. A rolling collision energy voltage was used for CID fragmentation for MS/MS spectra acquisitions. Mass was dynamically excluded for 22 s (Yu et al., 2019 (link)).

The fractionated samples were dissolved in 10 μL of 2% in 0.1% formic acid solution, and 500 ng of each fraction was loaded onto a nano-LC 1D plus system (Eksigent, Framingham, MA, USA) consisting of an in-house C₁₈ resin (5 µm Proteo 100 Å; Phenomenex Inc., Torrance, CA, USA) and a capillary column (ID 75 µm, OD 150 µm; Molex, Lisle, IL, USA). Elution was conducted using a gradient liquid chromatography method (5–25% acetonitrile for 90 min) and analyzed with an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) in positive ion mode at the Mass Spectrometry Convergence Research Center. The m/z data collection range was set at m/z 300–1800, and higher-energy collisional dissociation collision mode was used for fragmentation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD023933.