Ltq obitrap velos mass spectrometer

The nanoAcquity Ultra Performance LC system (Waters Corporation, Milford, USA) was used for peptide separation equipped with a C18 reversed-phase microcapillary trapping (Maisch, Ammerbuch-Entringen, Germany) as described previously [53 (link)]. A total of 90 samples (30 samples conducted in triplicate) were loaded and eluted for 40 min using a 5–40% CAN fraction-optimized nonlinear gradient in 0.1% formic acid. Eluted peptides were analyzed using a LTQ Obitrap Velos mass spectrometer (Thermo Fisher Scientific) as previously described [51 (link)]. The eluted peptides were electrosprayed with a distally applied spray voltage of 2.0 kV. The mass spectrometry analysis was carried out in a data-dependent manner. Survey scans were performed at a resolution of 30,000 at target values of 1,000,000 ions in the Orbitrap analyzer with maximum allowed fill times of 150 ms over a mass range of 300–1,600 m/z. Finally, the 20 most intense precursor ions were chosen for MS/MS fragmentation by collision-induced fragmentation. The normalized collision energy for the MS/MS was set to 30%, and the transfer tube temperature was maintained at 220 °C. Exclusion of precursor ion masses over a time window of 30 s was used to suppress repeated fragmentation of peaks.

The UPLC system (nanoAcquity Ultra Performance LC, Waters Corporation, Milford, MA, USA) was used for peptide separation. The UPLC column is a 75-μm inner diameter (id) ×15-cm-long fused-ailica capillary tube packed with RepoSil-Pur C₁₈-AQ 1.9 μm resin (Dr. Maisch, Ammerbuch-Entringen, Germany). Samples were loaded at 0.3 μL/min and eluted for 40 min using a 5–40% ACN fraction-optimized nonlinear gradient in 0.1% formic acid. Eluted peptides were analyzed using a LTQ-Obitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Survey scans were performed at a resolution of 30,000 at target values of 1,000,000 ions in the Orbitrap analyzer with maximum allowed fill times of 150 ms over a mass range of 300–1,600 m/z. The 20 most intense precursor ions were chosen for collision-induced fragmentation. A total of 5,000 ions were accumulated over a fill time of 25 ms and fragmented by wideband activation on each scan. Exclusion of precursor ion masses over a time window of 30 s was used to suppress repeated fragmentation of peaks21 (link)22 (link).