A neutralizing epitope on the SD1 domain of SARS-CoV-2 Spike targeted following infection and vaccination. Seow et al
Data S1: Deuterium uptake values for the 321 Spike peptides whose HDX was followed. Data S2: Deuterium uptake plots for peptides whose HDX was followed.
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Prior to conducting HDX-MS experiments, peptides were identified by digesting undeuterated Spike using the same protocol and identical LC gradient as detailed below and performing MSE analysis with a Synapt G2-Si mass spectrometer (Waters), applying collision energy ramping from 20-30 kV. Sodium iodide was used for calibration and Leucine Enkephalin was applied for mass accuracy correction. MSE runs were analyzed with ProteinLynx Global Server (PLGS) 3.0 (Waters) and peptides identified in 3 out of 4 runs, with at least 0.2 fragments per amino acid (at least 2 fragments in total) and mass error below 10 ppm were selected in DynamX 3.0 (Waters). Deuterium labelling was performed at two different temperatures, at 20 and 0 oC. HDX samples at 20 oC were labelled with LEAP PAL system Automation technology (Trajan), directly coupled to a nanoACQUITY UPLC. The exchange reactions were initiated by 6-fold dilution into deuterated PBS buffer (pHread 7.6), yielding to 83.3% of final deuterium content in the reaction mixture. The reaction was allowed to proceed for 15 s, 1min, 10min and 100min at 20 oC. Samples were quenched by 1:1 dilution into cold (0 oC) 100 mM phosphate buffer containing 4 M Urea and 0.5 M TCEP (final pHread 2.3), held for 30s at 0 oC and directly injected into the LC system. HDX samples at 0 oC were manually labelled on ice. The exchange reactions were initiated by 6-fold dilution in ice-cold deuterated PBS buffer and allowed to proceed for 20 s on ice (corresponding to approximately 2s at 20 oC). Samples were quenched 1:1 with ice-cold quench buffer, held for 30s on ice, snap-frozen in liquid nitrogen and kept at -80 oC until LC-MS analysis. Frozen samples labelled on ice were quickly thawed and injected into the LC system. Triplicates were performed. A maximally labelled sample was performed by labelling Spike in 6 M deuterated Urea in D2O and 20 mM TCEP, resulting in a final deuterium content as for the other labelled samples. This control was quenched after 6 hours by 1:1 dilution with ice-cold 100 mM phosphate buffer (final pHread 2.3), held for 30 s on ice, snap-frozen in liquid nitrogen and kept at -80 oC until LC-MS analysis. Protein samples, each containing 22.5 pmol of Spike (monomer), were quickly on-line digested at 20 oC into a dual protease column (Pepsin-Type XIII protease) and trapped/desalted for 3 min at 200 μL/min and at 0 oC through an Acquity BEH C18 1.7 μm VanGuard pre-column with Solvent A (0.23% formic acid in MilliQ water, pH 2.5). Peptides were eluted into an Acquity UPLC BEH C18 1.7 μm analytical column with a linear gradient raising from 8 to 40% of Solvent B (0.23% formic acid in acetonitrile) at a flow rate of 40 μL/min and at 0 oC. Then, peptides went through electrospray ionization in positive mode and underwent MS analysis with ion mobility separation. Data were analyzed with DynamX 3.0 and statistical analysis was performed with Deuteros 2.0 (Lau et al., 2021), applying 99.9% of confidence interval.