Xevo g2 mass spectrometer

An HDX MS experiment was performed as described
previously.^{37 (link)} Briefly, deuterium labeling
was initiated with a 20-fold dilution into D₂O buffer (50
mM sodium phosphate, pH 7.4, 100 mM NaCl) of a pre-equilibrated (30
min) aliquot of IGF2BP1 with or without CuB stock solution. After
0.25, 1, 10, 20, 60, and 240 min of labeling, the reaction was quenched
with the addition of quenching buffer (37.5% hydrochloric acid). Samples
were then injected and online-digested using a Waters Enzymate BEH
pepsin column (2.1 × 30 mm, 5 μm). The peptides were trapped
and desalted on a VanGuard Precolumn trap (Acquity UPLC BEH C18, 1.7
μm) for 3 min, eluted from the trap using 15% acetonitrile at
a flow rate of 100 μL/min, and then separated using an Acquity
UPLC BEH C18, 1.7 μm, 1.0 × 100 mm column. All mass spectra
data were acquired using a Waters Xevo G2 mass spectrometer. Peptides
were identified using ProteinLynx Global Server (PLGS) 3.0.2. Relative
deuterium levels of all peptides were calculated by subtracting the
mass of the undeuterated control sample from that of the deuterium-labeled
sample. All mass spectra data were processed using DynamX 3.0. Deuterium
levels were not corrected for back exchange and thus are reported
as relative.

Deuterium labeling was initiated with a 20-fold dilution into D₂O buffer (100 mmol/L phosphate, pH 7.0) of 3CL^pro protein (1 mg/mL), 3CL^pro (1 mg/mL) with schaftoside (1 mmol/L), PL^pro protein (1 mg/mL), or PL^pro (1 mg/mL) with schaftoside (1 mmol/L). After 0.25, 0.5, and 10 min of labeling, the labeling reaction was quenched with the addition of quenching buffer (100 mmol/L phosphate, 4 mol/L GdHCl, 0.5 mol/L TCEP, pH 2.0). Samples were then injected and online digested using a Waters ENZYMATE BEH pepsin column (2.1 mm × 30 mm, 5 μm). The peptides were trapped and desalted on a VanGuard Pre-Column trap (ACQUITY UPLC BEH C18, 1.7 μm) for 3 min, eluted from the trap using 15% acetonitrile at a flow rate of 100 μL/min, and then separated using an ACQUITY UPLC BEH C18 column (1.0 mm × 100 mm, 1.7 μm). All mass spectra were acquired on a Waters Xevo G2 mass spectrometer. Peptides from an unlabeled protein were identified using ProteinLynx Global Server (PLGS) searches of a protein database including 3CL^pro and PL^pro sequences only. Relative deuterium levels for each peptide were calculated by subtracting the mass of the undeuterated control sample from that of the deuterium-labeled sample. All mass spectra were processed using DynamX 3.0 (Waters Corporation). Deuterium levels were not corrected for back exchange and thus reported as relative.