Ultimate 3000 rslc nano uphlc system

Online nanoLC-MS/MS analyses were performed using an Ultimate 3000 RSLC Nano-UPHLC system (Thermo Scientific, USA) coupled to a nanospray Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific). Ten microliters of the peptide extract were loaded on a 300 µm ID × 5 mm PepMap C18 precolumn (Thermo Scientific) at a flow rate of 20 µL/min. After 5 min desalting, peptides were separated on a 75 µm ID × 25 cm C18 Acclaim PepMap® RSLC column (Thermo Scientific) with a 4–40% linear gradient of solvent B (0.1% formic acid in 80% ACN) in 108 min. The separation flow rate was set at 300 nL/min. The mass spectrometer operated in positive ion mode at a 1.8 kV needle voltage. Data were acquired using Xcalibur 3.1 software in a data-dependent mode. MS scans (m/z 350–1600) were recorded at the resolution of R = 70,000 (@ m/z 200) and an AGC target of 3 × 106 ions collected within 100 ms. Dynamic exclusion was set to 30 s and top 12 ions were selected from fragmentation in HCD mode. MS/MS scans with a target value of 1 × 105 ions were collected with a maximum fill time of 100 ms and a resolution of R = 17,500. In addition, only + 2 and + 3 charged ions were selected for fragmentation. The other settings were as follows: no sheath and no auxiliary gas flow, heated capillary temperature 200 °C, normalized HCD collision energy of 27%, and an isolation width of 2 m/z.

Online nanoLC-MS/MS analyses were performed using an Ultimate 3000 RSLC Nano-UPHLC system (Thermo Scientific, USA) coupled to a nanospray Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific, USA). 500 ng of each peptide extract was loaded on a 300 μm ID x 5 mm PepMap C₁₈ precolumn (Thermo Scientific, USA) at a flow rate of 10 μl min^-1. After a 3 min desalting step, peptides were separated on a 75 μm ID x 25 cm C₁₈ Acclaim PepMap RSLC column (Thermo Scientific, USA) with a 4–40% linear gradient of solvent B (0.1% formic acid in 80% ACN) in 108 min. The separation flow rate was set at 300 nl min^-1. The mass spectrometer operated in positive ion mode at a 1.8 kV needle voltage. Data was acquired using Xcalibur 3.1 software in a data-dependent mode. MS scans (m/z 300–1600) were recorded at a resolution of R = 70,000 (m/z 200) and an AGC target of 3 × 10⁶ ions collected within 100 ms. Dynamic exclusion was set to 30 s and top 12 ions were selected from fragmentation in HCD mode. MS/MS scans with a target value of 10⁵ ions were collected with a maximum fill time of 100 ms and a resolution of R = 17,500. Additionally, only +2 and +3 charged ions were selected for fragmentation. Other settings were as follows: no sheath and no auxiliary gas flow, heated capillary temperature, 200°C; normalized HCD collision energy of 27 eV and an isolation width of 2 m/z.