Nanoease symmetry c18 trapping column

Using an Acquity M-class nanoLC system (Waters, Milford, MA, USA), 5 µL of the sample was loaded at 15 μL/min for 3 min onto a nanoEase Symmetry C18 trapping column (180 μm × 20 mm) before being washed onto a PicoFrit column (75 μm ID × 350 mm; New Objective, Woburn, MA, USA) packed with SP-120-1.7-ODS-BIO resin (1.7 μm, Osaka Soda Co., Tokyo, Japan) heated to 45 °C at 300 nL/min. Peptides were eluted from the column and into the source of a Q Exactive Plus mass spectrometer (Thermo Scientific, Carlsbad, CA, USA) using the following program: 5–30% MS buffer B (98% acetonitrile + 0.2% formic acid) over 90 min, 30–80% MS buffer B over 3 min, 80% MS buffer B for 2 min, 80–5% for 3 min. The eluting peptides were ionised at 2400 V. A data-dependant MS/MS (dd-MS2) experiment was performed, with a survey scan of 350–1500 Da performed at 70,000 resolution for peptides of charge state 2+ or higher with an AGC target of 3 × 10⁶ and maximum injection time of 50 ms. The top 12 peptides were selected fragmented in the HCD cell using an isolation window of 1.4 m/z, an AGC target of 1 × 10⁵ and maximum injection time of 100 ms. Fragments were scanned in the Orbitrap analyser at 17,500 resolution, and the product ion fragment were masses measured over a mass range of 120–2000 Da. The mass of the precursor peptide was then excluded for 30 s.