Orbitrap velos pro

Analysis was performed on Orbitrap Velos Pro mass spectrometer using Xcalibur coupled with an Agilent 1200 G1311 Quaternary HPLC system. In detail, peptides were eluted and separated using an analytical column (100 μm × 15 cm, capillary filled with Jupiter 4 μm, 90 Å, Phenomenex), using a 10 min buffer gradient ranging from 0 to 10% buffer B, followed by a 139 min gradient from 10 to 50% buffer B and 10 min gradient from 50 to 100% buffer B at a flow rate of 250 μL per min utilizing a 1×1000 split flow (buffer A: 0.1% formic acid, 5% acetonitrile; buffer B: 0.1% formic acid, 80% acetonitrile). Data-dependent FTMS acquisition was in positive ion mode for 3 h. A full scan (300–2000m/z) was performed with a resolution of 60,000 followed by top 10 CID-MS2 ion trap scans. Dynamic exclusion was set for 20 s.

Fractions were run on a Thermo Fisher Orbitrap Velos Pro coupled with an Agilent NanoLC system (Agilent, Santa Clara, CA, USA) over a 60-min gradient. The LC columns (15 cm × 75 μm ID, Zorbax 300SB-C18) were purchased from Agilent. The samples were analyzed with a 60-minute linear gradient (0–35% acetonitrile with 0.1% formic acid), and data were acquired in a data-dependent manner, in which MS/MS fragmentation is performed on the top 10 most intense peaks of every full MS scan. RAW files were converted into.mgf files using MSConvert (from ProteoWizard)^{57 (link)}.

Samples were analyzed by a Thermo Orbitrap Velos Pro mass spectrometer coupled with an Agilent nanoflow LC system. Peptides were eluted on Agilent Zorbax 300SB-C18 column (3.5 um, 0.075 × 150 mm) via 60 min gradient LC program where mobile phase B increases from 7% to 40%. Mobile phase A: water with 0.1% FA; mobile phase B: ACN with 0.1%FA. Data were acquired by a DDA method (data-dependent acquisition); higher-energy collisional dissociation fragmentation was performed for the top-12 peaks in each MS scan.