Comprehensive HeLa Proteome Profiling

LC-MS instrumentation consisted of a breadboard Evosep One coupled to an LTQ Orbitrap for the more than 2000 HeLa injection experiment, and the Evosep One production version coupled to an Q Exactive HF-X Orbitrap (Thermo Fisher Scientific) for all other experiments. Purified peptides were separated on the HPLC columns with 3 μm Reprosil-Pur C18 beads (Dr. Maisch, Ammerbuch, Germany) and dimensions indicated below in Fig. 6B. On the LTQ Orbitrap MS, data were acquired with a Top6 data dependent shotgun method and with a Top12 method for the Q Exactive HF-X instrument. On the Q Exactive HF-X Orbitrap, the target value for the full scan MS spectra was 3 × 10⁶ charges in the 300–1650 m/z range with a maximum injection time of 50 ms and a resolution of 60,000 at m/z 200. Fragmentation of precursor ions was performed by higher-energy C-trap dissociation (HCD) with a normalized collision energy of 27 eV (24 (link)). MS/MS scans were performed at a resolution of 15,000 at m/z 200 with an ion target value of 5 × 10⁴ and a maximum injection time of 25 ms. Dynamic exclusion was set to 15 s to avoid repeated sequencing of identical peptides.