Peptide Analysis by Nano LC-MS/MS

Peptides were analyzed in an Dionex UltiMate 3000 RSLC nano System coupled on-line to Q Exactive Orbitrap High Field Hybrid Quadrupole Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, United States) as described previously (Aryal et al., 2018 (link); Mohallem and Aryal, 2020 (link)). Briefly, reverse phase peptide separation was accomplished using a trap column (300 μm ID × 5 mm) packed with 5 mm 100 Å PepMap C18 medium coupled to a 50-cm long × 75 μm inner diameter analytical column packed with 2 μm 100 Å PepMap C18 silica (Thermo Fisher Scientific). The column temperature was maintained at 50°C. Sample was loaded to the trap column at a flow rate of 5 μL/min and eluted from the analytical column at a flow rate of 300 nL/min using a 120-min LC gradient. The column was washed and equilibrated by using three 30 min LC gradient before injecting next sample. Precursor ion (MS1) scans were collected at a resolution of 120,000 and MS/MS scans at a resolution of 15,000 at 200 m/z in data dependent acquisition mode.