Aeris c18 material

The digestion extracts were analyzed by reversed phase high performance liquid chromatography (HPLC) using Waters NanoAcquity pumps and autosampler (Waters, Milford, MA) and a ThermoFisher Orbitrap Elite mass spectrometer (ThermoFisher Scientific, Waltham, MA) using a nano flow configuration. A 20 mm x 180 micron column packed with five micron Symmetry C18 material (Waters, Milford, MA) using a flow rate of 15 µL/min for 3 min was used to trap and wash peptides. These were then eluted onto the analytical column which was a self-packed with 3.6 micron Aeris C18 material (Phenomenex, Torence, CA) in a fritted 20 cm x 75 micron fused silica tubing pulled to a five micron tip. The gradient was isocratic 1% A Buffer for 1 min 250 nL/min with increasing B buffer concentrations to 15% B at 20.5 min, 27% B at 31 min and 40% B at 36 min. The column was washed with high percent B and re-equilibrated between analytical runs for a total cycle time of approximately 53 min. Buffer A consisted of 1% formic acid in water and buffer B consisted of 1% formic acid in acetonitrile.

The digestion extracts were analyzed by reverse-phase high-performance liquid chromatography (HPLC) using Waters NanoAcquity pumps and autosampler and a ThermoFisher Orbitrap Elite mass spectrometer using a nano flow configuration. A 20 mm × 180 μm column packed with 5 μm Symmetry C18 material (Waters) using a flow rate of 15 μl per min for 3 min was used to trap and wash peptides. These were then eluted onto the analytical column which was self-packed with 3.6 μm Aeris C18 material (Phenomenex) in a fritted 20 cm × 75 μm fused silica tubing pulled to a 5 μm tip. Elution was carried out with a gradient of isocratic 1% Buffer A (1% formic acid in H₂O) for 1 min (250 nL min⁻¹), followed by increasing Buffer B (1% formic acid in acetonitrile) concentrations to 15% B at 20.5 min, 27% B at 31 min and 40% B at 36 min. The column was washed with high percent B and re-equilibrated between analytical runs for a total cycle time of approximately 53 min.

Retention time calibration peptides (iRT-Kit, Biognosys, Schlieren, Switzerland) were spiked into each sample at 75× dilution of the iRT stock solution. SWATH–MS was carried out on a 5600+ triple-TOF mass spectrometer coupled to an Ekspert NanoLC 415 system (Eksigent, AB Sciex, Dublin, CA, USA) equipped with an in-house packed emitter tip column filled with 2.6 µm Aeris C18 material (Phenomenex, Torrance, CA, USA) on a length of 20 cm. Peptides were separated over a 120-minute gradient using a binary solvent system (solvent A: 1% ACN, 0.1% FA in water, solvent B: 90% ACN, 0.1% FA in water). To generate spectral libraries, the mass spectrometer was run in data-dependent acquisition (DDA) mode (technical details in Supplementary Methods). SWATH–MS spectra were acquired over a 2-hour gradient using variable window width for precursor ion selection (technical details in Supplementary Methods). Each sample was injected three times, and the median ion intensity was calculated across the three SWATH–MS runs.