Easy nlc 1200 instrument

One microgram of glycopeptides was separated on an EASY-nLC™ 1200 instrument (Thermo Fisher Scientific, Germany) with the use of Acclaim PepMap100 precolumn (2 cm, 75 µm i.d., 3 µm) and Acclaim PepMap100 separating column (50 cm, 75 µm i.d., 3 µm) operating at 300 nL/min. The mobile phase A (0.1% FA) and mobile phase B (0.1% FA / 80% ACN) with a flow rate of 200 nL/min. The parameters used for intact glycopeptides analysis were the same as previously described [22 (link), 23 (link)].

The HLA-I immunopeptidome was analyzed using an EASY-nLC 1200 instrument (Thermo Fisher Scientific, California, United States) equipped with a self-packed capillary column (75 μm i.d. × 20 cm, 1.9 μm C₁₈ reversed-phase fused silica) coupled to an Orbitrap Exploris 480 (Thermo Fisher Scientific, California, United States). The gradient was comprised of an increasing Buffer B (Buffer A: 0.1% FA in water; Buffer B: 0.1% FA in 100% ACN) from 5 to 10% for 4 min, 10 to 30% for 56 min, 30 to 95% for 6 min and holding for 2 min. Full MS scans ranged from 300 to 1600 m/z with a resolution of 60,000. The maximum injection time was 50 ms, and the normalized automatic gain control (AGC) target was set at 300% for the remaining settings. The MS₂ scan was configured to collect fragmentation for charge state of 2 to 6, using high-energy collision dissociation (HCD) with normalized collision energy of 27%, the resolution for MS₂ was set at 15,000, AGC target was set at 100%, and the maximum injection time was 40 ms. A dynamic exclusion time of 15 s was applied. Theoretical retention time were generated using DeepLC (https://iomics.ugent.be/deeplc/).