Pepmap c18 column

LC–MS/MS was performed in a Dionex UltiMateTM 3000 RSLCnano coupled to a LTQ Orbitrap XL mass spectrometer via a nanospray ion source (Thermo Fisher Scientific, Auckland, New Zealand). Peptides were fractioned on a PepMap C18 column (3 μm, 300 Å, 75 μm × 15 cm; ThermoFisher Scientific, Auckland, New Zealand) on a 350 min gradient from 0 to 80% ACN in 0.1% FA at a constant flow rate of 300 nL/min and eluted into the Orbitrap via a PicoTip emitter (360 × 20 μm; New Objective, Littleton, MA, USA) at a voltage set to 1.8 kV through a transfer tube of 25 μm inner diameter [31 (link),32 (link),33 (link)]. The six most intense peptide ions from the MS scan were selected and fragmented using collision-induced dissociation (normalised collision energy, 35%; activation Q, 0.250; and activation time, 30 ms) for MS/MS scans. Dynamic exclusion was used with the following settings: repeat count, 2; repeat duration, 30 s; exclusion list size, 500; exclusion duration, 90 s [32 (link)]. The spectra was acquired using Xcalibur (version 2.1.0 SP1, Thermo Fisher Scientific, Auckland, New Zealand).

All synthetic peptides were obtained from Genscript as a library service and crude purity. We measured the peptide libraries and HLA-peptides by LC-MS on an Ultimate 3000 RSLCnano System coupled with an Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific). Peptides were loaded onto the analytical column (PepMap C18 column, 2 µm particle size, 75 µm × 50 cm; Thermo Scientific) and eluted in a 60 min linear gradient from 3% to 25% ACN in 1% DMSO/0.1% formic acid at a flow rate of 250 nl/min. Peptides were introduced to the mass spectrometer using an EasySpray source at 2000 V and 45˚C, and the transfer tube temperature was set to 305˚C. Mass spectrometry (MS) detection was performed with a resolution of 120,000 for full MS (320-1600 m/z scan range) and AGC target of 300,000. A full-MS1 scan (120,000 resolution, 60 ms accumulation time, AGC 3×10⁶) was followed by 20 data-dependent MS2 scans (60,000 resolution, 120 ms accumulation time, AGC 5×10⁵), with an isolation width of 1.6 m/z and normalized HCD energy of 25%.

Each gel lane was excised into three or five equal pieces, which were destained with 50% 100 mM ammonium bicarbonate/50% acetonitrile (ACN). Proteins in the gels were reduced with 10 mM dithiothreitol, then alkylated with 55 mM iodoacetamide. Trypsin (20 ng) was added to each of the gel pieces followed by incubation overnight at 37°C. Peptide extraction was carried out in 5% formic acid (FA).
LC-MS analysis was on an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher) coupled to an Ultimate 3000 RSLCnano LC system. Peptides were resuspended in 0.1% trifluoracetic acid (TFA) and were then loaded onto a 100 μm × 2 cm PepMap C18 trap (100 Å, 5 μm) separated on a 75 μm x 50 cm PepMap C18 column (100 Å, 2 μm) (both from Thermo Fisher) using a linear gradient of 4 to 55% B in 65 min (solvent A: 0.1% FA/98% H₂O, 2% ACN, solvent B: 0.1%FA/80%, ACN/20%H₂O). The instrument was controlled by the Xcalibur software with a standard CID top six data dependent acquisition method. The resolution of Full MS survey was set at 15 000. The parent ion's isolation width was set at 2.0 Da, and the normalized collision energy at 35.0, activation Q at 0.25, activation time 30 ms and the lock mass at 445.120030 m/z.

Approximately 300 μg proteins from the main roots of TKS were reduced and alkylated by dithiothreitol and iodoacetamide, then digested by trypsin. Enzymic hydrolysates were separated using a Pepmap C18 column (Thermo Fisher, USA) following a 65 min 5%–35% organic gradient by an UltiMate 3000 instrument (Thermo Scientific, Rockford, USA). Fifty washed fractions were collected in 1.5 mL/min tubes. Fractions were dehydrated and merged into 15 fractions, and these samples were subjected to a high-resolution mass spectrometer Triple TOF 6600 system (AB SCIEX). Peptides were automatically selected by ProGroupTM algorithm and ProteinPilot™ software V5.0 (AB SCIEX) to calculate the error factor (EF), reporter peak area and p value as described [30 (link)]. A protein database was established by the target protein sequences (https://academic.oup.com/nsr) for the corresponding species. Protein with an unused score > 1.3 (confidence ≥ 95%) was considered as a positive identification. Finally, an in-house BlastP search of the UniProt database was performed for each protein to identify its homologues and potential functions.

Each fraction was resuspended in 50 μL 1% acetonitrile/1% formic acid. 5 μL was analyzed by LC-MS (HCD for MS/MS) with a Dionex RSLCnano HPLC coupled to a Velos Pro OrbiTrap mass spectrometer (Thermo Scientific) using a 2h gradient. Peptides were resolved using 75 μm x 25 cm PepMap C18 column (Thermo Scientific).

Mass analysis was performed using an EASY-nLC^TM 1200 system connected to a Thermo Orbitrap Fusion Lumos Tribrid Mass Spectrometer (ThermoFisher) equipped with a nano spray interface (New Objective). Peptide mixtures were loaded onto a 75-μm ID, 25 cm length PepMap C18 column (ThermoFisher) packed with 2 μm particles, pore size −100 Å and separated using a segmented gradient in 90 min from 5 to 45% solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nl/min. Solvent A was 0.1% formic acid in water. The mass spectrometer was operated in the data-dependent mode. Briefly, survey scans of peptide precursors from 350 to 1600 m/z were performed at 120 K resolution with a 2 × 10⁵ ion count target. Fragmentation (MS2) spectra were acquired in the Orbitrap at 60 K resolution. Precursor ions with 3–8 positive charges were selected for fragmentation with dynamic exclusion of 30 secs and isolation window of 1.6 Th. Additional MS2 settings included an automatic gain control target of 50,000 ions and a maximum injection time of 120 ms. The normalized collision energy was set to 30% for HCD scans. MS1 and MS2 scans were acquired in the Orbitrap.

Samples were analysed on an Orbitrap Fusion instrument on-line with an Ultimate 3000 RSLC nano UHPLC system (Thermo Fisher). Samples were resuspended in 10 µL 5% DMSO/1% TFA and all sample was injected. Trapping solvent was 0.1% TFA, analytical solvent A was 0.1% FA, solvent B was ACN with 0.1% FA. Samples were loaded onto a trapping column (300 µm x 5 mm PepMap cartridge trap (Thermo Fisher)) at 10 µL/min for 5 min at 60 degrees. Samples were then separated on a 75 cm x 75 µm i.d. 2 µm particle size PepMap C18 column (Thermo Fisher) at 55 degrees. The gradient was 3–10% B over 10 min, 10–35% B over 155 min, 35–45% B over 9 min followed by a wash at 95% B for 5 min and requilibration at 3% B. Eluted peptides were introduced by electrospray to the MS by applying 2.1kV to a stainless-steel emitter (5 cm x 30 µm (PepSep)). During the gradient elution, mass spectra were acquired with the parameters detailed in Figure 1—figure supplement 2 using Tune v3.3 and Xcalibur v4.3 (Thermo Fisher).