Acquity uplc peptide beh c18 nanoacquity column

After PNGase F/H₂¹⁸O treatment, peptide samples were analyzed on a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific) equipped with a nanoAcquity UPLC system (Waters) and a Triversa Nanomate (Advion, Ithaca, NY). For chromatographic separation, a nanoACQUITY UPLC Symmetry C18 Trap Column (100 Å, 5 μm; 180 μm × 20 mm, Waters) column was used for trapping, and an ACQUITY UPLC Peptide BEH C18 nanoACQUITY Column (130 Å, 1.7 μm; 150 μm × 100 mm, Waters) column was used for separation. The peptide trapping step was performed at 4 μL/min for 4 min with 1% acetonitrile and 0.1% formic acid (Solvent A). Following the trapping step, peptides were separated on the analytical column according to the following conditions: 0–1 min: 2% B, 1–3 min: 2–5% B, 3–43 min: 5–40% B (Solvent B: 99% acetonitrile and 0.1% formic acid). MS scans were acquired with the following settings: 70 000 resolution @ m/z 400, scan range m/z 370–1880, 1 μscan/MS, AGC target 1e6, and a maximum injection time of 100 ms. MS2 scans were acquired with the following settings: 17 500 resolution at m/z 400, AGC target of 5e5, maximum injection time of 60 ms, isolation window of 2.0 m/z, isolation offset of 0.4 m/z, normalized collision energy (NCE) of 27%, exclusion of charge states 1 and >8, underfill ratio of 1.2%, and dynamic exclusion of 8 s. Profile data were recorded for MS and MS2 scans.