Easy spray c18 lc column

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. 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 EDT 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 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 90 s to prevent repetitive selection of peptides [70 (link)].

Each sample was split into two volumes and subsequently analyzed on an EASY‐Spray C18 LC column (75 μm × 120 mm, 3 μm) (Thermo Fisher Scientific). A Q Exactive mass spectrometer (Thermo Fisher Scientific) combined with an EASY‐nLC 1000 system (Nano HPLC, Thermo Fisher Scientific) was used to analyze the samples. Ten microliters of each sample was separated using mobile phase A (99.9% ultrapure water, 0.1% formic acid) and mobile phase B (99.9% acetonitrile, 0.1% formic acid), with a gradient of 5% to 95% mobile phase B for 126 min, at a flow rate of 0.3 μl/min. Then, the eluate was analyzed using the Q Exactive mass spectrometer.
The MS data were acquired in high sensitivity mode, using the following analytical parameters: ion source EASY‐Spray, spray voltage 2.3 kV; capillary temperature, 320℃; full scan automatic gain control (AGC), target 3E6; resolution, 70,000 (full width at half maximum, fwhm); full scan maximum injection time, 20 ms; and scan range, 300 − 1,800 m/z. The MS/MS spectrum parameters were as follows: resolution, 17,500 (fwhm); AGC target, 1E5; maximum injection time, 120 ms; and intensity threshold, 8.30E3. A total of 30 data‐dependent MS/MS scans were acquired for each full scan. Precursor ions were further fragmented in a collision cell using normalized collision energy of 32%, and the fragments were quantitated using an Orbitrap.

Samples were resuspended in 10.5 μL 0.1% FA, and 1.5 μL of the suspension injected for HPLCESI-MS/MS analysis. Data acquisition was performed in positive ion mode on a ThermoScientific Orbitrap Fusion Lumos tribrid mass spectrometer fitted with an EASY-SpraySource (Thermo Scientific, San Jose, CA). NanoLC was performed using a ThermoScientific UltiMate 3000 RSLCnano System with an EASY Spray C18 LC column (ThermoScientific, 50 cm × 75 μm inner diameter, packed with PepMap RSLC C18 material, 2 μm, cat. # ES803): loading phase for 15 min at 0.300 μl/min; linear gradient of 1–34% Buffer B in 119 min at 0.220 μl/min, followed by a step to 95% Buffer B over 4 min at 0.220 μL/min, hold 5 min at 0.250 μl/min, and then a step to 1% Buffer B over 5 min at 0.250μl/min and a final hold for 10 in (total run 159 min); Buffer A = 0.1% FA; Buffer B = 0.1%FA in 80% ACN. Spectra were collected using XCalibur, version 2.3 (ThermoFisherScientific). Precursor scans were acquired in the Orbitrap at 120,000 resolution on a mass range from 375 to 1575 Th. Precursors were isolated with an isolation width of 1.6 Th and subjected to higher energy collisional dissociation. MS/MS scans were acquired in the ion trap on the m/z range of 120 to 2000 Th with a fill time of 35 ms.

HPLC-ESI-MS/MS was performed in positive ion mode on a Thermo Scientific Orbitrap Fusion Lumos tribrid mass spectrometer fitted with an EASY-Spray Source (Thermo Scientific, San Jose, CA, USA) as previously described [63 (link)]. In brief, nanoflow liquid chromatography was performed without a trap column using a Thermo Scientific UltiMate 3000 RSLCnano System with an EASY Spray C18 LC column (Thermo Scientific, 50 cm × 75 μm inner diameter, packed with PepMap RSLC C18 material, 2 µm, cat. # ES803); loading phase for 15 min at 0.300 mL/min; mobile phase, linear gradient of 1–34% Buffer B in 119 min at 0.220 mL/min, followed by a step to 95% Buffer B over 4 min at 0.220 mL/min, hold 5 min at 0.250 mL/min, and then a step to 1% Buffer B over 5 min at 0.250 mL/min and a final hold for 10 min (total run 159 min); Buffer A = 0.1% FA/H₂O; Buffer B = 0.1% FA in 80% ACN. All solvents were liquid chromatography mass spectrometry grade. Spectra were acquired using XCalibur, version 2.3 (Thermo Scientific). A “top speed” data-dependent MS/MS analysis was performed. Dynamic exclusion was enabled with a repeat count of 1, a repeat duration of 30 sec, and an exclusion duration of 60 s.