Easy nlc 1000 nano uplc chromatography

The MS analyses were performed using an Easy-nLC 1000 nano-UPLC chromatography (Thermo Scientific) interfaced with a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific) with a nano-electrospray ion source. The sample was loaded onto an Acclaim PepMap100 C18 reverse-phase trap column (100 μm×2 cm) with nanoViper fittings (Thermo Scientific) connected to the C18-reversed-phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3 μm resin) in buffer A (0.1% Formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min controlled by IntelliFlow technology. The mass spectrometer was operated in the positive ion mode. MS data were acquired using a data-dependent top10 method that dynamically selected the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD fragmentation. The automatic gain control (AGC) target was set to 1e6, and the maximum inject time was set to 50 ms. The dynamic exclusion duration was 60.0 s. Survey scans were acquired at a resolution of 70,000 at 200 m/z, the resolution for HCD spectra was set to 17,500 at 200 m/z, and the isolation window was 2 m/z. The normalized collision energy was 30 eV, and the underfill ratio was defined as 0.1%. The instrument was run with the peptide recognition mode enabled.

The MS analysis were performed using an Easy-nLC 1000 nano-UPLC chromatography (Thermo Scientific) interfaced with a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific) with a nano-electrospray ion source. The sample was loaded onto an Acclaim PepMap100 C18 reverse-phase trap column (100 μm × 2 cm) with nanoViper fittings (Thermo Scientific) connected to the C18-reversed-phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3 μm resin) in buffer A (0.1% Formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min controlled by IntelliFlow technology. The mass spectrometer was operated in the positive ion mode. MS data were acquired using a data-dependent top10 method that dynamically selected the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD fragmentation. The automatic gain control (AGC) target was set to 1e6, and the maximum inject time was set to 50 ms. The dynamic exclusion duration was 60.0 s. Survey scans were acquired at a resolution of 70,000 at 200 m/z, the resolution for HCD spectra was set to 17,500 at 200 m/z, and the isolation window was 2 m/z. The normalized collision energy was 30 eV, and the underfill ratio was defined as 0.1%. The instrument was run with the peptide recognition mode enabled.