Nanospray flex esi

Mobile phase A (100% water, 0.1% formic acid) and B solution (80% acetonitrile, 0.1% formic acid) were prepared. The lyophilized powder was dissolved in 10 μL of solution A, centrifuged at 15,000 rpm for 20 min at four ℃, and 1 μg of the supernatant was injected into a home-made C18 Nano-Trap column (2 cm×75 μm, three μm). Peptides were separated in a home-made analyticalcolumn (15 cm×150 μm, 1.9 μm) using linear gradient elution, as listed in Supplemental Table 1. The isolated peptides were analyzed by the Q Exactive series mass spectrometer (Thermo Fisher), with ion source of Nanospray Flex™ ESI , spray voltage of 2.3 kV, and ion transport capillary temperature of 320°C. Full scan range from m/z 350 to 1500 with resolution of 60000 (at m/z 200), an automatic gain control (AGC) target value was 3×10 6, and a maximum ion injection time was 20 ms. The top 20 40 precursors of the highest abundant in the full scan were selected and fragmented by higher-energy collisional dissociation (HCD) and analyzed in MS/MS, where resolution was 15000 (at m/z 200), the automatic gain control (AGC) target value was 5×10 4 , the maximum ion injection time was 45 ms, a normalized collision energy was set as 27%, and intensity threshold was 2.2×10 4 . The dynamic exclusion parameter was 20 s. The raw data of M.S. detection was named as ".raw".

Mobile phase A (100% water, 0.1% formic acid) and B solution (80% acetonitrile, 0.1% formic acid) were prepared. The lyophilized powder was dissolved in 10 μL of solution A, centrifuged at 15,000 rpm for 20 min at four ℃, and 1 μg of the supernatant was injected into a home-made C18 Nano-Trap column (2 cm×75 μm, three μm). Peptides were separated in a home-made analyticalcolumn (15 cm×150 μm, 1.9 μm) using linear gradient elution, as listed in Supplemental Table 1. The isolated peptides were analyzed by the Q Exactive series mass spectrometer (Thermo Fisher), with ion source of Nanospray Flex™ ESI , spray voltage of 2.3 kV, and ion transport capillary temperature of 320°C. Full scan range from m/z 350 to 1500 with resolution of 60000 (at m/z 200), an automatic gain control (AGC) target value was 3×10 6, and a maximum ion injection time was 20 ms. The top 20 40 precursors of the highest abundant in the full scan were selected and fragmented by higher-energy collisional dissociation (HCD) and analyzed in MS/MS, where resolution was 15000 (at m/z 200), the automatic gain control (AGC) target value was 5×10 4 , the maximum ion injection time was 45 ms, a normalized collision energy was set as 27%, and intensity threshold was 2.2×10 4 . The dynamic exclusion parameter was 20 s. The raw data of M.S. detection was named as ".raw".