Ultimate system 3000 nano lc system

Peptides were separated by using an Ultimate System 3000 Nano-LC system (Thermo Fisher Scientific, Bremen, Germany) equipped with a 75 μm inside diameter (ID) and 25 cm length C18 Acclaim PepMap Nano-LC column (Thermo Fisher Scientific, San Jose, CA, USA) and packed with 2 μm particles with a pore size of 100 Å. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 100% acetonitrile with 0.1% formic acid. A segmented gradient from 2% to 35% solvent B for 90 min, at a flow rate of 300 mL/min, and a column temperature of 35 °C were used. Intact peptide mass spectra and fragmentation spectra were acquired on a Thermo Scientific ™ Orbitrap Fusion ™ Lumos ™ Tribrid ™ Mass Spectrometer (Thermo Fisher Scientific, UK). Mass spectrometry analysis was performed in a data-dependent mode with Full-MS (externally calibrated to a mass accuracy of <5 ppm, and a resolution of 120,000 at m/z = 200), followed by high-energy collision activated dissociation (HCD)-MS/MS of the most intense ions in 3 s. HCD-MS/MS (resolution of 15,000) was used to fragment multiply charged ions (charge state 2-7) within a 1.4 Da isolation window at a normalized collision energy of 32 eV. Automatic gain control (AGC) target at 5e⁵ and 5e⁴ was set for MS and MS/MS analysis, respectively, with previously selected ions dynamically excluded for 180 s.

High-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) was performed on an Orbitrap Fusion Lumos Tribrid quadrupole-ion trap mass spectrometer (Thermo Fisher Scientific) in the Instrumentation Center of National Taiwan University. Peptides were separated on an Ultimate System 3000 NanoLC System (Thermo Fisher Scientific). Peptide mixtures were loaded onto a 75 μm inner diameter (ID), 25 cm length C18 Acclaim PepMap NanoLC column (Thermo Scientific) packed with 2 μm particles with a pore size of 100 Å. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 100% acetonitrile with 0.1% formic acid. A segmented gradient was set over 90 min from 2% to 35% solvent B at a flow rate of 300 nl/min. Mass spectrometry analysis was performed in a data-dependent mode with full-MS (externally calibrated to a mass accuracy of < 5 ppm, and a resolution of 120,000 at m/z = 200), followed by high-energy collision activated dissociation (HCD)-MS/MS of the most intense ions in 3 s. HCD-MS/MS (resolution of 15,000) was used to fragment multiply charged ions within a 1.4 Da isolation window at a normalized collision energy of 32. An automatic gain control (AGC) target at 5e5 and 5e4 was set for MS and MS/MS analysis, respectively, with previously selected ions dynamically excluded for 180 s. The max injection time was 50 ms.

Peptides were separated using an Ultimate System 3000 nano-LC system (Thermo Fisher Scientific, Bremen, Germany) equipped with a 75 μm ID, 25 cm length C18 Acclaim PepMap Nano-LC column (Thermo Fisher Scientific, San Jose, CA, USA) packed with 2 μm particles with a pore size of 100 Å. Mobile phase A and B were 0.1% formic acid in water and 100% acetonitrile with 0.1% formic acid, respectively. A segmented gradient from 2% to 35% solvent B in 90 min at a flow rate of 300 nL/min was conducted, and we maintained the column temperature at 35 °C for LC analysis. Intact peptide mass spectra and fragmentation spectra were acquired on a Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer (Thermo Fisher Scientific, Hemel Hempstead, UK). Mass spectrometry (MS) analysis was performed in data-dependent mode with Full-MS (externally calibrated to a mass accuracy of <5 ppm and a resolution of 120,000 at m/z = 200), followed by high-energy collision-activated dissociation (HCD)-MS/MS of the most intense ions in 3 s. HCD-MS/MS (resolution of 15,000) was used to fragment multiply charged ions (charge state 2–7) within a 1.4 Da isolation window at a normalized collision energy of 32 eV. An AGC target at 5 × 10⁵ and 5 × 10⁴ was set for MS and MS/MS analysis, respectively, with previously selected ions dynamically excluded for 180 s. Max. injection time was 50 ms.