Reversed phase columns were prepared in-house. Briefly, a 75–360 μm inner-outer diameter bare-fused silica capillary, with a laser pulled electrospray tip, was packed with 1.7 μm diameter, 130 Å pore size, Bridged Ethylene Hybrid C18 particles (Waters) to a final length of 35 cm. The column was installed on a nanoAcquity UPLC (Waters) using a stainless steel ultra-high pressure union formatted for 360 μm outer diameter columns (IDEX) and heated to 60 °C for all runs. Mobile phase buffer A was composed of water, 0.2% formic acid, and 5% DMSO. Mobile phase B was composed of acetonitrile, 0.2% formic acid, and 5% DMSO. Samples were loaded onto the column for 12 min at 0.35 μl/min. Mobile phase B increases to 4% in the first 0.1 min then to 12% B at 32 min, 22% B at 60 min, and 30% B at 70 min, followed by a 5 min wash at 70% B and a 20 min re-equilibration at 0%B.
Eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a Thermo Orbitrap Fusion (Q-OT-qIT, Thermo). Survey scans of peptide precursors from 300 to 1500 m/z were performed at 60K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS was performed by isolation at 0.7 Th with the quadrupole, HCD fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion trap. The MS2 ion count target was set to 104 and the max injection time was 35 ms. Only those precursors with charge state 2–6 were sampled for MS2. The dynamic exclusion duration was set to 45 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 5 s cycles, meaning the instrument would continuously perform MS2 events until the list of nonexcluded precursors diminishes to zero or 5 s, whichever is shorter. Elite runs were performed with Survey scans of peptide precursors from 300 to 1500 m/z 60K resolution (at 200 m/z) with a 1 × 106 ion count target. Tandem MS was performed by isolation at 1.8 Th with the ion-trap, CAD fragmentation with normalized collision energy of 35, and rapid scan MS analysis in the ion trap. The data dependent top 20 precursors were selected for MS2. MS2 ion count target was set to 5 × 103 and the max injection time was 125 ms. Only those precursors with charge state +2 or higher were sampled for MS2. The dynamic exclusion duration was set to 40 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on.
Eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a Thermo Orbitrap Fusion (Q-OT-qIT, Thermo). Survey scans of peptide precursors from 300 to 1500 m/z were performed at 60K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS was performed by isolation at 0.7 Th with the quadrupole, HCD fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion trap. The MS2 ion count target was set to 104 and the max injection time was 35 ms. Only those precursors with charge state 2–6 were sampled for MS2. The dynamic exclusion duration was set to 45 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 5 s cycles, meaning the instrument would continuously perform MS2 events until the list of nonexcluded precursors diminishes to zero or 5 s, whichever is shorter. Elite runs were performed with Survey scans of peptide precursors from 300 to 1500 m/z 60K resolution (at 200 m/z) with a 1 × 106 ion count target. Tandem MS was performed by isolation at 1.8 Th with the ion-trap, CAD fragmentation with normalized collision energy of 35, and rapid scan MS analysis in the ion trap. The data dependent top 20 precursors were selected for MS2. MS2 ion count target was set to 5 × 103 and the max injection time was 125 ms. Only those precursors with charge state +2 or higher were sampled for MS2. The dynamic exclusion duration was set to 40 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on.
