Ekspert nanolc 425 system

Each sample was analysed on a SCIEX TripleTof 6600 mass spectrometer coupled in line with an Eksigent ekspert nano LC 425 system running in microflow as described previously [31 (link)] with minor modifications. In brief, 6 μg (3 uL) of sample was injected via trap/elute. The following linear gradients were used (5 uL/min): mobile phase B (acetonitrile + 0.1% formic acid) over mobile phase A (0.1% formic acid) as follows: SWATH (57 min run) increasing from 3 to 30% over 38 min, 30 to 40% over 5 min, 40 to 80% over 2 min; IDA (87 min run) increasing from 3 to 30% over 68 min, 30 to 40% over 5 min, 40 to 80% over 2 min followed by wash and re-equilibration.

Chromatographic separation was performed either on an Ekspert NanoLC 425 system (Eksigent/SCIEX) for combined nano and micro flow analysis, or a nanoACQUITY system (Waters) for microflow-only sample series. In nano flow, the NanoLC 425 system was equipped with a nanolitre flow module, and samples were first loaded onto a trap column (Chrom XP C18–3µm, 0.12 nm, 0.35 × 0.5 mm) by isocratically running the system at a flow rate of 5 µL/min for 6 min with 0.1% formic acid (FA) in water. Peptides were then eluted onto the analytical column (3C18-CL-120, 3 µm, 0.12 nm, 0.075 × 150 mm, Eksigent) and separated on a linear gradient of 2–30% 0.1% FA in acetonitrile (ACN) in 25 min. For microlitre flow rate chromatography, the same system was equipped with a low microlitre flow module (1–5 µL/min) or high microlitre flow module (5–10 µL/min) and set up for direct injection onto an analytical column (3C18-CL-120, 3 µm, 120 Å, 0.3 × 150 mm, Eksigent). Separation was performed on a linear gradient of 2–30% 0.1% FA in ACN in 25 min. For microlitre flow rate chromatography on the nanoACQUITY system, the sample manager was set up in direct injection mode and equipped with a Triart C18 column (0.12 nm, 3 µm, 0.3 mm × 250 mm, YMC). After injecting samples onto the analytical column, peptides were separated on linear gradients detailed in Suppl. Table 2.