Reprosil pur c18 aq 1.9 m silica beads

Nanoflow LC‐MS analysis of tryptic peptides was performed on a quadrupole Orbitrap mass spectrometer (Q Exactive HF‐X, Thermo) coupled to an EASY‐nLC1200 system (Thermo) via a nano‐electrospray ion source. Approx. 0.25 µg of sample was loaded on a 50‐cm HPLC‐column (75‐μm inner diameter, New Objective, USA; in‐house packed using ReproSil‐Pur C18‐AQ 1.9‐µm silica beads; Dr Maisch GmbH, Germany). Peptides were separated using a linear gradient from 5 to 60% solvent B (80% CAN; 0.1% formic acid) in solvent A (0.1% formic acid). Run duration was 100 min (whole proteome) or 60 min (AP‐MS). Column temperature was maintained at 60°C. Peptide detection occurred using “top‐15” (whole proteome) or “top‐10” (AP‐MS) data‐dependent mode, collecting MS spectra in the Orbitrap mass analyzer (60,000 resolution, 300–1,650 m/z range) with an automatic gain control (AGC) target of 3 × 10⁶ and a maximum ion injection time of 25 ms. The most intense ions from the full scan were isolated with an isolation width of 1.4 m/z. Following higher‐energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 27%, MS/MS spectra were collected in the Orbitrap (15,000 resolution) with an AGC target of 1 × 10⁵ and a maximum ion injection time of 28 ms (whole proteome) or 50 ms (AP‐MS). Precursor dynamic exclusion was enabled with a duration of 30 s.