Tune v3

Samples were analysed on an Orbitrap Fusion instrument on-line with an Ultimate 3000 RSLC nano UHPLC system (Thermo Fisher). Samples were resuspended in 10 µL 5% DMSO/1% TFA and all sample was injected. Trapping solvent was 0.1% TFA, analytical solvent A was 0.1% FA, solvent B was ACN with 0.1% FA. Samples were loaded onto a trapping column (300 µm x 5 mm PepMap cartridge trap (Thermo Fisher)) at 10 µL/min for 5 min at 60 degrees. Samples were then separated on a 75 cm x 75 µm i.d. 2 µm particle size PepMap C18 column (Thermo Fisher) at 55 degrees. The gradient was 3–10% B over 10 min, 10–35% B over 155 min, 35–45% B over 9 min followed by a wash at 95% B for 5 min and requilibration at 3% B. Eluted peptides were introduced by electrospray to the MS by applying 2.1kV to a stainless-steel emitter (5 cm x 30 µm (PepSep)). During the gradient elution, mass spectra were acquired with the parameters detailed in Figure 1—figure supplement 2 using Tune v3.3 and Xcalibur v4.3 (Thermo Fisher).

Samples were analysed on an Orbitrap Fusion instrument online with an Ultimate 3000 RSLC nano UHPLC system (Thermo Fisher Scientific). Samples were resuspended in 10 μl 5% DMSO/1% TFA. A 5 μl of each fraction was Injected for TMT experiments, the whole sample was injected for AP‐MS experiments. Trapping solvent was 0.1% TFA, analytical solvent A was 0.1% formic acid, solvent B was ACN with 0.1% formic acid. Samples were loaded onto a trapping column (300 μm × 5 mm PepMap cartridge trap (Thermo Fisher Scientific)) at 10 μl/min for 5 min. Samples were then separated on a 75 cm × 75 μm i.d. A 2 μm particle size PepMap C18 column (Thermo Fisher Scientific). The gradient was 3–10% B over 10 min, 10–35% B over 155 min, 35–45% B over 9 min followed by a wash at 95% B for 5 min and re‐equilibration at 3% B. Eluted peptides were introduced by electrospray to the MS by applying 2.1 kV to a stainless‐steel emitter (5 cm × 30 μm (PepSep)). During the gradient elution, mass spectra were acquired with the parameters detailed in Appendix Supplementary Materials and Methods (A) using Tune v3.3 and Xcalibur v4.3 (Thermo Fisher Scientific).