Reprosil pur c18 aq 1.9 m beads

Each sample was analyzed as technical duplicate on a LC–ESI–MS-MS quadrupole Orbitrap QExactive-HF mass spectrometer (Thermo Fisher Scientific, Milan, Italy). Separation of peptides was achieved on a linear gradient from 93% solvent A (2% ACN, 0.1% formic acid) to 60% solvent B (80% acetonitrile, 0.1% formic acid) over 110 min, and from 60% to 100% solvent B in 10 min at a constant flow rate of 0.25 µL/min on UHPLC Easy-nLC 1000 (Thermo Fischer Scientific, Milan, Italy) connected to a 23 cm fused-silica emitter of 75 µm ID (New Objective, Inc. Woburn, MA, USA), packed in-house with ReproSil-Pur C18-AQ 1.9 µm beads (Dr Maisch Gmbh, Ammerbuch, Germany) using a high-pressure bomb loader (Proxeon, Odense, Denmark). MS data were acquired using a data-dependent top 20 method for HCD fragmentation. Survey full scan MS spectra (300–1650 Th) were acquired in the Orbitrap with resolution 60,000, AGC target 3e6, IT 20 ms. For HCD spectra, resolution was set to 15,000 at m/z 200, AGC target 1e5, IT 80 ms; NCE 28%, dynamic exclusion 20 s and isolation width 1.2 m/z [63 (link)].

Samples were analyzed as described in^{81 (link)} with few exceptions. In particular, 4 µL of peptide mixture from each sample were analyzed on a nLC–ESI–MS/MS quadrupole Orbitrap QExactive-HF mass spectrometer (Thermo Fisher Scientific). Peptides separation was achieved on a linear gradient from 95% solvent A (2% ACN, 0.1% formic acid) to 60% solvent B (80% acetonitrile, 0.1% formic acid) over 48 min, and from 60 to 100% solvent B in 2 min at flow rate of 0.25 µL/min on a UHPLC Easy-nLC 1000 (Thermo Fischer Scientific) connected to a 23 cm fused-silica emitter of 75 µm inner diameter (New Objective, Inc. Woburn, MA, USA), packed in-house with ReproSil-Pur C18-AQ 1.9 µm beads (Dr. Maisch GmbH, Ammerbuch, Germany). MS data were acquired using a data-dependent top 15 method for HCD fragmentation. Survey full scan MS spectra (300–1800 Th) were acquired in the Orbitrap with 60,000 resolution, AGC target 3^e6, IT 20 ms. For HCD spectra, resolution was set to 15,000 at m/z 200, AGC target 1^{e5 (link)}, IT 160 ms; NCE 28% and isolation width 1.4 m/z.