Stainless steel emitter

Samples were analyzed on an Orbitrap Fusion instrument on-line with an UltiMate 3000 RSLCnano UHPLC system (Thermo Fisher). Samples were resuspended in 10 μl of 5% dimethyl sulfoxide–1% trifluoroacetic acid (TFA), and 5 μl of each fraction was injected. The trapping solvent was 0.1% TFA, the analytical solvent A was 0.1% formic acid, and solvent B was acetonitrile with 0.1% formic acid. Samples were loaded onto a trapping column (300 μm × 5 mm PepMap cartridge trap; Thermo Fisher) at 10 μl/min for 5 min. Samples were then separated on a PepMap C₁₈ column (50 cm × 75 μm i.d., 2-μm particle size; Thermo Fisher). The gradient was 3 to 10% solvent B over 10 min, 10 to 35% solvent B over 155 min, 35 to 45% solvent B over 9 min, followed by a wash with 95% solvent B for 5 min and reequilibration with 3% solvent B. Eluted peptides were introduced by electrospray to the MS by applying 2.1 kV to a stainless-steel emitter (5 cm × 30 μm; Thermo Fisher). During the gradient elution, MS1 spectra were acquired in the Orbitrap, and the collision-induced dissociation (CID)-MS2 was acquired in the ion trap. Synchronous precursor selection-isolated MS2 fragment ions were further fragmented using higher-energy collisional dissociation to liberate reporter ions which were acquired in the Orbitrap (MS3).

The eluate from the analytical column was sprayed via stainless steel emitters (Thermo) at a source voltage of 2.6 kV towards the orifice of the mass spectrometer; a transfer capillary heated to 275°C. The Orbitrap Velos was set to data‐dependent acquisition in positive ion mode, automatically selecting the top 10 most intense precursor ions from the preceding full MS (MS1) spectrum with an isolation width of 2.0 Th for fragmentation using higher‐energy collisional dissociation (HCD) at 30% normalised collision energy and subsequent identification by MS/MS (MS2). MS1 (360–1,300 m/z) and MS2 (precursor‐dependent m/z range, starting at m/z 100) spectra were acquired in the Orbitrap using a resolution of 30,000 and 7,500 at m/z 400, with an automatic gain control (AGC) target value of 1 × 10⁶ and 3 × 10⁴ charges and a maximum injection time of 100 and 200 ms, respectively. Dynamic exclusion was set to 60 s.

LC-MS/MS analysis was performed as previously published^{1 (link)} with following changes. An aliquot of 500 ng of each sample was injected in a nano–HPLC (Dionex Ultimate 3000) equipped with a C18, 5 µm, 100 Å, 20 × 0.1 mm, enrichment column and an Acclaim PepMap RSLC nanocolumn (C18, 2 µm, 100 Å, 500 × 0.075 mm) (Thermo Fisher Scientific). Samples were concentrated on the enrichment column for 6 min at a flow rate of 5 µl/min with 0.1% formic acid as isocratic solvent. Separation was carried out on the nanocolumn at a flow rate of 250 nl/min at 60 °C using the following gradient, where solvent A is 0.1% formic acid in water and solvent B is acetonitrile containing 0.1% formic acid: 0–6 min: 4% B; 6–264 min: 4–25% B; 264–274 min: 25–95% B, 274–289 min: 95% B; 289.1–304 min: 4% B; The sample was ionized in the nanospray source equipped with stainless steel emitters (Thermo Fisher Scientific, Vienna, Austria) and analyzed in a Thermo Orbitrap velos pro mass spectrometer in positive ion mode by alternating full scan MS (m/z 300 to 2000, 60000 resolution) and MS/MS by collision-induced dissociation (CID) of the 10 most intense peaks in the ion trap with dynamic exclusion enabled.