Integrafrit capillary

Phosphopeptide-enriched samples were analyzed by LC-MS/MS on an Easy-nLC 1000 (ThermoFisher) coupled to an LTQ-Orbitrap Fusion mass spectrometer (ThermoFisher) operated in positive ion mode. The LC system, configured in a vented format consisted of a fused-silica nanospray needle (PicoTip emitter, 50μm ID x 20cm, New Objective) packed in-house with ReproSil-Pur C18-AQ, 3μm and a trap (IntegraFrit Capillary, 100μm ID x 2 cm, New Objective) containing the same resin as in the analytical column with mobile phases of 0.1% FA in water (A) and 0.1% FA in MeCN (B). The peptide sample was diluted in 20 µL of 0.1% FA, 3% MeCN, and 8.5μL was loaded onto the column and separated over 210 minutes at a flow rate of 300 nL/min with a gradient from 5 to 7% B for 2 minutes, 7 to 35% B for 150 minutes, 35 to 50% B for 1 minute, hold 50% B for 9 minutes, 50 to 95% B for 2 minutes, hold 95% B for 7 minutes, 95 to 5% B for 1 minute, re-equilibrate at 5% B for 38 minutes. A spray voltage of 2000 V was applied to the nanospray tip. MS/MS analysis occurred over a 3 second cycle time consisting of 1 full scan MS from 350–1500 m/z at resolution 120,000 followed by data dependent MS/MS scans using HCD activation with 27% normalized collision energy of the most abundant ions. Selected ions were dynamically excluded for 45 seconds after a repeat count of 1.

Phosphopeptide-enriched samples were analyzed by LC-MS/MS on an Easy-nLC 1000 (ThermoFisher) coupled to an LTQ-Orbitrap Fusion mass spectrometer (ThermoFisher) operated in positive ion mode. The LC system, configured in a vented format consisted of a fused-silica nanospray needle (PicoTip emitter, 50μm ID × 20cm, New Objective) packed in-house with ReproSil-Pur C18-AQ, 3μm and a trap (IntegraFrit Capillary, 100μm ID × 2 cm, New Objective) containing the same resin as in the analytical column with mobile phases of 0.1% FA in water (A) and 0.1% FA in MeCN (B). The peptide sample was diluted in 20 μL of 0.1% FA, 3% MeCN, and 8.5 μL was loaded onto the column and separated over 210 minutes at a flow rate of 300 nL/min with a gradient from 5 to 7% B for 2 minutes, 7 to 35% B for 150 minutes, 35 to 50% B for 1 minute, hold 50% B for 9 minutes, 50 to 95% B for 2 minutes, hold 95% B for 7 minutes, 95 to 5% B for 1 minute, re-equilibrate at 5% B for 38 minutes. A spray voltage of 2000 V was applied to the nanospray tip. MS/MS analysis occurred over a 3 second cycle time consisting of 1 full scan MS from 350–1500 m/z at resolution 120,000 followed by data dependent MS/MS scans using HCD activation with 27% normalized collision energy of the most abundant ions. Selected ions were dynamically excluded for 45 seconds after a repeat count of 1.