Hydrogen/Deuterium Exchange Mass Spectrometry.
A DHR-PAL system (Trajan) was used for sample preparation, with sample
tray at 20°C, quench tray at 4°C, valve chamber & pre-chiller at 4°C,
and digestion chamber at 8°C. Purified spike ectodomain (0.4 μg/μL) was
mixed 1:5 (v/v) with PBS prepared using H2O
(equilibration buffer, measured pH 7.22) or D2O
(labeling buffer, measured pH 7.56). Labeling times were 0, 60, 240, and
960 sec with 3 technical replicates at each time. Samples were quenched
with an equal volume of 2 M urea 0.5 M TCEP pH 2.5 and held at 4°C for 2
min. An UltiMate3000 UPLC system (binary nano pump and loading pump,
Thermo Scientific) was used for subsequent online sample handling.
Automated valve switching passed the quenched sample over a 2.1 × 20 mm
Nepenthesin-2 / Pepsin mixed digestion column (AffiPro, CZ) at 100
μL/min H2O 0.1% HCOOH for 2 minutes, trapping the
resulting peptides on a 2.1 × 5 mm Fully Porous C18 guard column
(Phenomenex, CA), then de-salted peptides at 300 μL/min for 4 minutes.
Peptides were eluted and resolved by a gradient from 13 to 65% mobile
phase B (95:5:0.1 CH3CN / H2O / HCOOH
over 23 min on a 1 × 100 mm Luna Omega 1.6 μm 100 Å C18 column
(Phenomenex, CA). A Tribrid Eclipse Orbitrap (OT) mass spectrometer
(Thermo Scientific) with HESI-2 electrospray ion source and high-flow
needle was operated in positive ion mode to detect peptides for 31
minutes. In all samples precursor scans of resolution 120K (atm/z 200) in the range 375-2000 m/z were acquired.For each MS scan, the top-10 abundant precursor features were selected
for data-dependent MS/MS scans, selecting for an intensity threshold of
30K counts, monoisotopic peptide precursors, charge states
2+ to 8+ with an isolation window of
1.2 m/z , and not repeating precursor ions more than twice within
15 sec. Precursors were fragmented with HCD at 28% normalized collision
energy (NCE) and centroid scanned in the OT with standard automatic gain
control (AGC) target, automatic injection time, scan range 120-2000m/z , and resolution 30K. If at least one of three selected
oxonium ions was detected (HexNAc 204.0867 m/z , HexNAc fragment
138.0545 m/z , or HexNAcHex 366.1396 m/z ) with 15 ppm mass
tolerance then EThcD OT-MS2 scans were acquired of that precursor ion.
Supplemental HCD was at 20% NCE with profile scans from 150-2000m/z at resolution 50K using custom AGC target (500%) and fill
time (90 msec). At the end of the analytical gradient, solvent
transitioned to 90% mobile phase B for 6 min, and halfway through that
time the analytical and trapping columns were put into back-flow washing
mode by automated valve switching. After re-equilibration of the
analytical column at 13% mobile phase B the injection cycle ended at 45
min. As recommended (42), the entire batch of control and heat denatured
samples (all time points and technical replicates) was randomized for
HDX-MS acquisition to minimize batch effects on interpreted differences
in protein state, labeling time, and replicates. To allow access to the
HDX data of this study, the HDX data summary table (Table S2) and the
HDX data table (Table S3) are included in the supporting information as
per consensus guidelines (42).