HDX-MS dataset for: "Glycan-induced structural activation softens the human papillomavirus capsid for entry through reduction of intercapsomere flexibility"

Hydrogen/deuterium exchange mass spectrometry dataset used in: Glycan-induced structural activation softens the human papillomavirus capsid for entry through reduction of intercapsomere flexibility. Yuzhen Feng*, Dominik van Bodegraven*, Alan Kádek*, Ignacio L.B. Munguira, Laura Soria-Martinez, Sarah Nentwich, Sreedeepa Saha, Florian Chardon, Daniel Kavan, Charlotte Uetrecht#, Mario Schelhaas#, Wouter H. Roos#. Nature Communications 10076 (2024). doi: 10.1038/s41467-024-54373-0

* - authors contributing equally

# - corresponding authors

Description:

Hydrogen/deuterium exchange mass spectrometry (HXMS) analysis of the effect of heparin on the conformational dynamics of human papillomavirus 16 pseudovirus (PsV).

Sample processing:

HPV16 PsV were prepared according to (Buck & Thompson: Current Protocols in Cell Biology 2007). In short, p16Shell and pClneo-EGFP were transfected into HEK293TT cells. After 48 h, cells were harvested and lysed followed by maturation of the virus particles for 24 h. For purification, the particles were purified using a CsCl step gradient (27 % w/V and 38.8 % w/V CsCl in 10 mM Tris-HCl pH 7.4, 207570 x g, 3 h 50 min, 4 °C) followed by dialysis in Float-A-Lyzer devices (1 mL, Spectra/Por) against a total of 3 L HPV virion buffer (1x PBS, 635 mM NaCl, 0.9 mM CaCl2, 0.5 mM MgCl2, 2.1 mM KCl, pH 7.4).

PsV were pre-incubated for 1 h either with or without heparin (H4784, Sigma-Aldrich) at room temperature. To initiate deuterium labelling the samples were 6-fold diluted with the virion buffer they were obtained in, only made of 99.9% D2O (150 mM NaCl, 4.8 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.9 mM CaCl2, 0.5 mM MgCl2, pD 7.2). This resulted in a final concentration of 0.5 µM L1 monomer in the form of PsV with or without 1 mg/ml heparin during deuterium labelling. The exchange reaction was left to proceed at room temperature until aliquots of 45 µl were removed at predetermined time points (1 min, 5 min, 15 min, 1 h and 4 h). In the aliquots, the exchange was immediately stopped by twofold dilution with ice-cold quench buffer (0.25 M glycine, 100 mM TCEP, 8 M urea, indicated pH 2.7), resulting in final pH 2.5. For samples with heparin, the quench buffer additionally contained 1 mg/ml protamine sulphate (P4020, Sigma-Aldrich). After 30 s incubation on ice, the samples were centrifuged at 10.000 x g for 1 min at 0 °C. Each supernatant was transferred to a fresh tube and flash frozen in liquid nitrogen. Low binding microtubes and low binding pipette tips (both Axygen) were used throughout for all handling of viral particles.

The frozen samples were quickly thawed and injected into a refrigerated (1°C) HPLC system (Infinity 1260, Agilent Technologies), through a porcine pepsin column (≥ 3200 units/mg, Sigma-Aldrich) in-house immobilized onto POROS-20AL perfusion resin (Thermo Scientific) as described previously (Wang et al.: Molecular & Cellular Proteomics 2002), which was kept at 4°C. Pepsin digestion was performed at isocratic 200 µl/min flow rate (0.4 % formic acid in water). After the digestion, peptides were online desalted for 3 min on a peptide microtrap (OPTI-TRAP, Optimize Technologies) and then eluted on a reversed-phase analytical column (ZORBAX 300SB-C18, 0.5 x 35 mm, 3.5 µm, 300Å, Agilent Technologies). There LC separation proceeded at 25 µl/min flow rate through an 8 min gradient of 8–30% solvent B, followed by a 3 min gradient of 30-90 % solvent B (solvent A: 0.4 % formic acid in water, solvent B: 0.4 % formic acid in acetonitrile). The outlet of the HPLC system was connected to an electrospray ionization (ESI) source of an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Scientific). The instrument was operated in positive ESI MS-only mode for deuterated samples, scan range 300-2000 m/z, using 4 microscans at resolving power setting 120,000. In a separate measurement on non-deuterated sample, the instrument was used in positive data-dependent ESI MS/MS mode with 30% HCD dissociation, 1 microscan and 240,000 resolving power setting for the identification of all peptides produced by non-specific pepsin cleavage.

In total 22 pmol and 50 pmol L1 protein were injected per MS and MS/MS analysis, respectively. To minimize sample carry-over on the protease column, two washing solutions were always injected between sample injections modified from Majumdar et al. 69 (wash solution 1: 5% acetonitrile, 5% isopropanol, 20% acetic acid; wash solution 2: 4 M Urea, 1 M glycine, pH 2.5). All HDX samples were analysed in technical triplicates, except for the 15 min time point for PsV without heparin, which was measured in duplicate.

Data processing:

Peptides were identified from the MS/MS data by the Andromeda search algorithm implemented in MaxQuant (version 1.6.5.0) using a custom protein database containing the sequences of HPV16 L1 and L2 proteins. Deuterium uptake for the identified peptides was calculated with DeutEx (in-house developed), manually inspected and the statistical significance of the observed differences in deuteration was evaluated by applying an unpaired two-tailed Student’s T-test with single pooled variance evaluated with alpha ≤ 0.05 using the Holm-Šidák correction for multiple comparisons in Prism 8.0.1 (GraphPad Software). The processed data were visualized using MSTools (https://peterslab.org/MSTools/, Kavan & Man: International Journal of Mass Spectrometry 2011) and open-source PyMol 2.6.0a0 (Schrödinger, Inc).

For ZENODO the datafiles were deposited as native Thermo .raw files (including instrumental parameters metadata) while all peaks in the spectra were additionally exported into plain m/z vs intensity .txt files per each scan in the LC-MS analysis as also used for the DeutEx HDX-MS processing.