C18 beads

The peptides were analyzed on an Orbitrap analyzer (Q-Exactive, ThermoFisher, San Jose, CA, USA) outfitted with a nanospray source and EASY-nLC nano-LC system (ThermoFisher, San Jose, CA, USA). a 75 μm × 50 cm PepMax RSLC EASY-Spray column filled with 2 μM C18 beads (ThermoFisher, SanJose, CA, USA) was used to load the peptide mixture at a pressure of 800 Bar. Peptides were then subjected to a stepwise gradient elution over 240 min at a rate of 250 nL/min (0–4% Acetonitrile containing 0.1% Formic Acid over 2 min; 4–28% Acetonitrile containing 0.1% Formic Acid over 226 min, 28–95% Acetonitrile containing 0.1% Formic Acid over 2 min, constant 95% Acetonitrile containing 0.1% Formic Acid for 10 min). In the Q-Exactive mass spectrometer (ThermoFisher, San Jose, CA, USA), one MS full scan (525–1600 m/z) was performed with an automatic gain control (AGC) target of 1 × 10⁶ maximum ion injection time of 120 ms and a resolution of 35,000 with subsequent 15 data-dependent MS/MS scans with a resolution of 35,000, an AGC target of 1 × 10⁶ , maximum ion time of 120 ms, and one microscan. The intensity threshold required to trigger a MS/MS scan was at an underfill ratio of 0.2%. In the higher energy collision dissociation (HCD) trap, normalized collision energy of 30 V was used for the fragmentation. The dynamic exclusion was applied with an exclusion period of 40 s [17 (link)].

Orbitrap analyzer (Q-Exactive, ThermoFisher, San Jose, CA) outfitted with a nanospray source and EASY-nLC nano-LC system (ThermoFisher, San Jose, CA). Lyophilized peptide mixtures were dissolved in 0.1% formic acid and loaded onto a 75 μm x 50 cm PepMax RSLC EASY-Spray column filled with 2 μM C18 beads (ThermoFisher San, Jose CA) at a pressure of 800 Bar. Peptides were eluted over 60 min at a rate of 250 nl/min using a 0% to 35% acetonitrile gradient in 0.1% formic acid. Peptides were introduced by nanoelectrospray into the Q-Exactive mass spectrometer (Thermo-Fisher). The instrument method consisted of one MS full scan (400–1500 m/z) in the Orbitrap mass analyzer with an automatic gain control target of 1e6, maximum ion injection time of 120 ms and a resolution of 70,000 followed by 10 data dependent MS/MS scans with a resolution of 17,500, an AGC target of 1e6, maximum ion time of 120 ms, and one microscan. The intensity threshold to trigger a MS/MS scan was set to 1.7e4. Fragmentation occurred in the HCD trap with normalized collision energy set to 27. The dynamic exclusion was applied using a setting of 10 s.

Peptide analysis was performed with the Q Exactive HF (Thermo Fisher Scientific, Waltham, MA, USA) as previously reported (Lyratzakis et al., 2021 (link)). For LC-MS/MS injection, 2.5 μg dried peptide fractions were dissolved in 2% acetonitrile with 0.1% formic acid, and subsequently loaded on reverse phase columns (trapping cartridge 5 µm C18-beads, L = 5 mm, inner diameter = 100 µm; Thermo Fisher Scientific, Waltham, MA, USA), analytical column (C18, 3 µm, 150 mm × 75 µm; Eksigent, Dublin, CA, USA). Eluted peptides were separated over a 78 min gradient of water (buffer A: water with 0.1% formic acid) and acetonitrile (buffer B: acetonitrile with 0.1% formic acid). Typically, gradients were ramped from 5% to 32% B in 70 min at flowrates of 600 nL min⁻¹. Electrospray voltage of 2.0 kV vs. the inlet of the mass spectrometer was used. The mass spectrometer was operated in data-dependent acquisition mode to automatically switch between Orbitrap-MS and ion trap acquisition. Survey of full-scan MS spectra (from m/z 300 to 1,400) were acquired in the Orbitrap with a resolution of R = 120,000. Target ions already selected for MS/MS were dynamically excluded for 18 s and the minimum intensity was 5,000.