Orbitrap fusion etd ms

Peptides were analyzed by LC (EASY-nLC 1000; Thermo Fisher Scientific, San Jose, CA, USA) combined with MS/MS (Orbitrap Fusion ETD MS; Thermo Fisher Scientific, San Jose, CA, USA) as described in the previous study [46 (link)] (Table S1). The peptides were loaded onto the LC system equipped with a trap column (Acclaim PepMap 100 C18 LC column, 3 µm, 75 µm ID × 20 mm; Thermo Fisher Scientific, San Jose, CA, USA) equilibrated with 0.1% formic acid and eluted with a linear acetonitrile gradient (0–35%) in 0.1% formic acid at a flow rate of 300 nL/min. The eluted peptides were loaded and separated on the column (EASY-Spray C18 LC column, 3 µm, 75 µm ID x 150 mm; Thermo Fisher Scientific, San Jose, CA, USA) with a spray voltage of 2 kV (Ion Transfer Tube temperature: 275 °C). The peptide ions were detected using MS in the data-dependent acquisition mode with the installed Xcalibur software (version 4.0; Thermo Fisher Scientific, San Jose, CA, USA). Full-scan mass spectra were acquired in the MS over 375–1500 m/z with a resolution of 120,000. The most intense precursor ions were selected for collision-induced fragmentation in the linear ion trap at a normalized collision energy of 35%. Dynamic exclusion was employed within 60 sec to prevent the repetitive selection of peptides.

The samples were then analyzed using a LC system (EASY-nLC 1000; Thermo Fisher Scientific) coupled to a MS (Orbitrap Fusion ETD MS; Thermo Fisher Scientific). The LC conditions as well as MS acquisition conditions are described in the previous study [64 (link)]. Briefly, the peptides were loaded onto the LC system equipped with a trap column (Acclaim PepMap 100 C18 LC column, 3 µm, 75 µm ID × 20 mm; Thermo Fisher Scientific) equilibrated with 0.1% formic acid and eluted with a linear acetonitrile gradient (0–35%) in 0.1% formic acid at a flow rate of 300 nL/min. The eluted peptides were loaded and separated on the column (EASY-Spray C18 LC column, 3 µm, 75 µm ID × 150 mm; Thermo Fisher Scientific) with a spray voltage of 2 kV (Ion Transfer Tube temperature: 275 °C). The peptide ions were detected using the MS with the installed Xcalibur software (version 4.0; Thermo Fisher Scientific).

The nano-liquid chromatography (LC) conditions as well as the mass spectrometry (MS) acquisition conditions are described in the previous study [60 (link)]. The peptides were loaded onto the LC system (EASY-nLC 1000; Thermo Fisher Scientific, San Jose, CA, USA) equipped with a trap column (Acclaim PepMap 100 C18 LC column, 3 µm, 75 µm ID × 20 mm; Thermo Fisher Scientific), equilibrated with 0.1% formic acid, and eluted with a linear acetonitrile gradient (0–35%) in 0.1% formic acid at a flow rate of 300 nL min⁻¹. The eluted peptides were loaded and separated on the column (EASY-Spray C18 LC column, 3 µm, 75 µm ID × 150 mm; Thermo Fisher Scientific) with a spray voltage of 2 kV (Ion Transfer Tube temperature: 275 °C). The peptide ions were detected using MS (Orbitrap Fusion ETD MS; Thermo Fisher Scientific) in the data-dependent acquisition mode with the installed Xcalibur software (version 4.0; Thermo Fisher Scientific). Full-scan mass spectra were acquired in the MS over 375–1500 m/z with a resolution of 120,000. The most intense precursor ions were selected for collision-induced fragmentation in the linear ion trap at a normalized collision energy of 35%. Dynamic exclusion was employed within 60 s to prevent repetitive selection of peptides.