Pepmap c18 easy spray lc column

An HLA peptides were separated by an Ultimate 3000 RSLC nano system (Thermo Scientific) using a PepMap C18 EASY-Spray LC column, 2 $\mu$ m particle size, 75 $\mu$ m × 75 cm column (Thermo Scientific) in buffer A (0.1% formic acid) and coupled online to an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific, United Kingdom) with a nanoelectrospray ion source. Peptides were eluted with a linear gradient of 3%−30% buffer B (ACN and 0.1% formic acid) at a flow rate of 300 nl/min over 110 min. Full scans were acquired in the Orbitrap analyzer using the Top Speed data dependent mode, preforming an MS scan every 3 s cycle, followed by higher-energy collision-induced dissociation (HCD) MS/MS scans. The MS spectra were acquired at resolution of 120 000 at 300 m/z, RF lens 60% and an automatic gain control ion target value of 4.0e5 for a maximum of 100 ms. The MS/MS resolution was 30 000 at 100 m/z. Higher-energy collisional dissociation (HCD) fragmentation was induced at an energy setting of 28 for peptides with a charge state of 2–4, whereas singly charged peptides were fragmented at an energy setting of 32 at lower priority. Fragments were analyzed in the Orbitrap at 30 000 resolution. Fragmented m/z values were dynamically excluded for 30 s.

HLA peptides were separated by an Ultimate 3000 RSLC nano system (Thermo Scientific) using a PepMap C18 EASY-Spray LC column, 2 μm particle size, 75 μm x 50 cm column (Thermo Scientific) in buffer A (0.1% Formic acid) and coupled on-line to an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific, UK) with a nano-electrospray ion source. Peptides were eluted with a linear gradient of 3%-30% buffer B (Acetonitrile and 0.1% Formic acid) at a flow rate of 300 nL/min over 110 minutes. Full scans were acquired in the Orbitrap analyser using the Top Speed data dependent mode, performing a MS scan every 3 second cycle, followed by higher energy collision-induced dissociation (HCD) MS/MS scans. MS spectra were acquired at resolution of 120,000 at 300 m/z, RF lens 60% and an automatic gain control (AGC) ion target value of 4.0e5 for a maximum of 100 ms. MS/MS resolution was 30,000 at 100 m/z. Higher-energy collisional dissociation (HCD) fragmentation was induced at an energy setting of 28 for peptides with a charge state of 2–4, while singly charged peptides were fragmented at an energy setting of 32 at lower priority. Fragments were analysed in the Orbitrap at 30,000 resolution. Fragmented m/z values were dynamically excluded for 30 seconds.