Q exactive mass spectrometer ms

The LC-20AD nano-high performance HPLC liquid system (Shimadzu, Kyoto, Japan) was used to suspend the peptide in buffer A according to the instructions, and the final concentration was about 0.5 μg/μL. 10 μL of supernatant with an automatic sampling system was aspirated and placed on a 2 cm C18 trapping column, and then eluted into a 10 cm C18 analytical column (inner diameter 75 μm). The samples were loaded at a rate of 8 μL/min for 4 min, then a concentration gradient of 2% to 35% of buffer B (98% ACN, 0.1% FA) was used to run at a flow rate of 300 nL/min for 44 min; then at 2 min the concentration of Buffer B was increased to 80% within 4 min, and finally, the concentration of Buffer B was reduced to 5% within 1 min. The peptides were separated by a nano-liquid chromatography system (HPLC), which was directly connected to the Q-EXACTIVE mass spectrometer (MS, ThermoFisher Scientific, San Jose, CA, USA), and the separated samples were analyzed by the Q-EXACTIVE mass spectrometer. For MS scanning, the m/z range of the primary MS full scan was 350–2000 u, and the m/z range of the secondary MS full scan was 100–1800 u.