Reprosil pur beads

Peptides were analyzed by LC-MS/MS using a Dionex UltiMate 3000 rapid separation nanoLC instrument and an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Inc., San Jose, CA). Samples were loaded onto the trap column, which was 150 μm by 3 cm, and in-house packed with 3-μm ReproSil-Pur beads. The analytical column was a 75-μm by 10.5-cm PicoChip column packed with 3-μm ReproSil-Pur beads (New Objective, Inc., Woburn, MA). The flow rate was kept at 300 nl/min. Solvent A was 0.1% formic acid (FA) in water and solvent B was 0.1% FA in ACN. The peptide was separated on a 120-min analytical gradient from 5% ACN/0.1% FA to 40% ACN/0.1% FA. MS¹ scans were acquired from 400 to 2,000 m/z at a 60,000 resolving power and with the automatic gain control (AGC) set to 1 × 10⁶ intensity. The 15 most abundant precursor ions in each MS¹ scan were selected for fragmentation by collision-induced dissociation (CID) at 35% normalized collision energy in the ion trap. Previously selected ions were dynamically excluded from reselection for 60 seconds.

Peptides were analyzed by LC-MS/MS using a Dionex UltiMate 3000 Rapid Separation LC (RSLC) systems and a linear ion trap—Orbitrap hybrid Elite mass spectrometer (Thermo Fisher Scientific Inc., San Jose, CA). Six-microliter peptide samples were loaded onto the trap column, which was 150 μm × 3 cm in-house packed with 3 μm ReproSil-Pur® beads (New Objective, Inc. Woburn, MA). The analytical column was a 75 μm × 10.5 cm PicoChip column packed with 3-μm ReproSil-Pur® beads. The flow rate was kept at 300 nL/min. Solvent A was 0.1% FA in water, and Solvent B was 0.1% FA in ACN. The peptide was separated on a 120-min analytical gradient from 5% ACN/0.1% FA to 40% ACN/0.1% FA. The mass spectrometer was operated in data-dependent mode. The source voltage was 2.40 kV, and the capillary temperature was 275 °C. MS1 scans were acquired from 400 to 2000 m/z at 60,000 resolving power and automatic gain control (AGC) set to 1 × 106. The top fifteen most abundant precursor ions in each MS1 scan were selected for fragmentation. Precursors were selected with an isolation width of 1 Da and fragmented by collision-induced dissociation (CID) at 35% normalized collision energy in the ion trap; previously selected ions were dynamically excluded from re-selection for 60 s. The MS2 AGC was set to 3 × 105.