Liquid chromatography

Peptides were separated by nanoLC using an UltiMate nanoRSLC UPLC and autosampler system (Dionex). Samples (2.5 µl) were concentrated and desalted on a micro C18 precolumn (300 µm×5 mm; Dionex) with H₂O/CH₃CN (98 : 2, 0.2 % trifluoroacetic acid) at 15 µl min⁻¹. After a 4 min wash, the pre-column was switched (Valco 10 port UPLC valve; Valco) into line with a fritless nano column (75 µm×~15 cm) containing C18AQ media (1.9μ, 120 Å; Dr Maisch). Peptides were eluted using a linear gradient of H₂O/CH₃CN (98 : 2, 0.1 % formic acid) to H₂O/CH₃CN (64 : 36, 0.1 % formic acid) at 200 nl min⁻¹ over 30 min. High voltage (2000 V) was applied to a low-volume titanium union (Valco) and the tip was positioned ~0.5 cm from the heated capillary (T=275 °C) of an Orbitrap Fusion Lumos (Thermo Electron Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the Fusion Lumos was operated in data-dependent acquisition mode (DDA).

A survey scan of m/z 350–1750 was acquired (resolution=120 000 at m/z 200, with an accumulation target value of 400 000 ions) and lockmass-enabled (m/z 445.12003). Data-dependent tandem MS analysis was performed using a top-speed approach (cycle time of 2 s). MS2 spectra were fragmented by high-energy collision dissociation (HCD; NCE=30) activation mode and the ion-trap was selected as the mass analyser. The intensity threshold for fragmentation was set to 25 000 units. A dynamic exclusion of 20 s was applied with a mass tolerance of 10 p.p.m.