Analysis of HLA ligands by LC–MS/MS

Isolated peptides were separated by reversed‐phase liquid chromatography (nano‐UHPLC, UltiMate 3,000 RSLCnano; ThermFisher) and analyzed in an online‐coupled Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher). Samples were analyzed in five technical replicates, and sample shares of 20% were trapped on a 75 μm × 2 cm trapping column (Acclaim PepMap RSLC; Thermo Fisher) at 4 μl/min for 5.75 min. Peptide separation was performed at 50 °C and a flow rate of 175 nl/min on a 50 μm × 25 cm separation column (Acclaim PepMap RSLC; Thermo Fisher) applying a gradient ranging from 2.4 to 32.0% of acetonitrile over the course of 90 min. Samples were analyzed on the Orbitrap Fusion Lumos implementing a top‐speed CID method with survey scans at 120 k resolution and fragment detection in the Orbitrap (OTMS2) at 60 k resolution. The mass range was limited to 400–650 m/z with precursors of charge states 2+ and 3+ eligible for fragmentation.