75 μm 25 cm c18 column

Peptides were resolved on a Thermo Scientific Dionex UltiMate 3000 RSLC system using a PepMap 75 μm × 25 cm C18 column with 2 μm particle size (100 Å pores), heated to 40°C. A final volume of 5 μl was injected, corresponding to 1 μg of total peptide, and separation was performed in a total run time of 90 min with a flow rate of 200 μl/min with mobile phases A: water/0.1% formic acid, and B: 80%ACN/0.1% formic acid. Gradient elution was performed from 10% to 8% B over 3 min, from 8% to 46% B over 66 min, and from 46 to 99% B over 3 min, and after holding at 99% B for 2 min, down to 2% B in 0.5 min followed by equilibration for 15 min.

Peptides were resolved on a Thermo Fisher Scientific Dionex UltiMate 3000 RSLC system using a PepMap 75 μm×25 cm C18 column with 2 μm particle size (100 Å pores), heated to 40°C. A 0.6 μg of total peptide amount was injected for each sample, and separation was performed in a total run time of 90 min with a flow rate of 200 μl/min with mobile phases A (water/0.1% formic acid) and B (80%ACN/0.1% formic acid). Gradient elution was performed from 10% to 8% B over 3 min, from 8% to 46% B over 66 min, and from 46 to 99% B over 3 min, and, after holding at 99% B for 2 min, down to 2% B in 0.5 min, followed by equilibration for 15 min. Peptides were directly eluted into an Orbitrap Exploris 480 instrument (Thermo Fisher Scientific, Bremen, Germany). Spray voltage was set at 1.8 kV, funnel radio frequency level at 45 and heated capillary temperature at 275°C. The full MS resolution was set to 60,000 at m/z 200 and full MS automatic gain control (AGC) target was 300% with an injection time set to Auto. Mass range was set to 350-1500. AGC target value for fragment spectra was set at 200% with a resolution of 15,000, and injection time was set to Standard and Top40. Intensity threshold was kept at 5E3. Isolation width was set at 1.6 m/z, and normalized collision energy was set at 30%.