Acclaim pepmap 100 c18 lc column

All samples were analyzed with a Thermo Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, San Jose, CA) equipped with a Thermo EASY-nanoLC system (Thermo Scientific, San Jose, CA) and a nanoelectrospray source. Five microliters of sample were injected into an Acclaim™ PepMap™ 100 C18 LC Column (Thermo Scientific, San Jose, CA) and separated with the Thermo EASY-nanoLC system loaded with a Thermo Scientific™ EASY-Spray™ C18 LC Column (Thermo Scientific, San Jose, CA) (2 μm, 75 μm × 50 cm). The samples were separated at a flow rate of 250 nl/min over 80 minutes with a gradient from 5% to 95% buffer B (99.9% Acetonitrile/0.1% formic acid). The raw data were collected continuously with a mass spectrometer in a data-dependent manner. A survey scan was recorded in the Orbitrap analyzer with a 60,000 resolution over a mass range between m/z 400–1600 Da and an automatic gain control (AGC) target at 4.0e⁵. Then, it was followed by the second stage of higher-energy collisional dissociation (HCD) MS/MS scans to the top 20 most intense ions from the survey scan with an AGC target of 1e³, a signal threshold of 1,000, auto scan range mode and 28% of HCD collision energy. Charge state was assigned to focus on ions that have a charge state of +2 and +3. Dynamic exclusion was enabled for 30 seconds repeat duration and 20 seconds exclusion duration with a repeat count of 3.

Details of this analysis have been described previously ((Wu et al., 2010 (link); Wu et al., 2011 (link))). The enriched glycopeptide samples were reconstituted with nanopure water and directly characterized using UltiMate^™ WPS-3000RS nanoLC 980 system coupled to the Nanospray Flex ion source of an Orbitrap Fusion Lumos Tribrid Mass Spectrometer system (Thermo Fisher Scientific, MA, United States). The analytes were separated on an Acclaim^™ PepMap^™ 100 C18 LC Column (3 μm, 0.075 mm × 150 mm, ThermoFisher Scientific). A binary gradient was applied using 0.1% (v/v) formic acid in (A) water and (B) 80% acetonitrile: 0–5 min, 4–4% (B); 5–133 min, 4–32% (B); 133–152 min, 32%–48% (B); 152–155 min, 48–100% (B); 155–170 min, 100–100% (B); 170–171 min, 100–4% (B); 171–180 min, 4–4% (B). The instrument was run in data-dependent mode with 1.8 kV spray voltage, 275°C ion transfer capillary temperature, and the acquisition was performed with the full MS scanned from 700 to 2000 in positive ionization mode. Stepped higher-energy C-trap dissociation (HCD) at 30 ± 10% was applied to obtain tandem MS/MS spectra with m/z values starting from 120.

Glycopeptide
measurements were carried out on an Ultimate 3000 RSLCnano UHPLC (Thermo
Fisher Scientific) coupled with a Q Exactive Plus Hybrid Quadrupole-Orbitrap
mass spectrometer (Thermo Fisher Scientific). The injection volume
was 1.0 μL. Chromatographic separation was achieved using a
75 μm × 500 mm Acclaim Pepmap 100 C18 LC column (Thermo
Fisher Scientific) operated at 50 °C and a flow rate of 300 nL
min^–1. Eluent A comprised water with 0.1% FA and
eluent B comprised acetonitrile with 0.1% FA. A gradient from 1 to
30% B over 75 min was applied, followed by an increase from 30 to
60% B in 15 min. 99% B was held for 10 min and equilibration was carried
out at 1% B for 35 min. The ion-source spray voltage was set to 1.5
kV, capillary temperature to 250 °C, in-source CID to 0, and
S-lens RF level to 60. For MS1, the Orbitrap mass analyzer m/z range was set to m/z 400–2000 with a resolution setting of
70 000 at m/z 200 and microscan
1, and the AGC target was 3 × 10⁶ with a maximum IT
of 100 ms. For MS/MS, the mass range was m/z 200–2000 with a resolution setting of 17 500
at m/z 200. The AGC target value
was 1 × 10⁵. The maximum injection time was set to
50 ms. The normalized collision energy (NCE) was 28. Microscans were
not averaged.

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.

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). The liquid chromatography (LC) conditions as well as the mass spectrometry (MS) acquisition conditions were described in the previous study [43 (link)].