Characterization of the binding of PPACK to Mpro by HDX-MS

Resulting peptic peptides were online trapped on a Acquity UPLC BEH C18 VanGuard Pre-column (130Å, 1.7µm, 2.1mmX5mm) and separated using a Acquity UPLC BEH C18 column (130Å, 1.7 µm, 1 mm X 100 mm) with a linear acetonitrile-0.1% (v/v) formic acid gradient from 5% to 60% in 10 min, at a flow rate of 60 μl/min. Mass spectra were acquired in the resolution mode (m/z range 50–2000). Unlabeled proteins were prepared as reference sample in the same manner as those that were labeled with deuterium.

Each sample was prepared in triplicate. Data analysis. Peptides that were generated from online pepsin digestion were identified from the unlabeled protein samples using Waters Protein Lynx Global Server 3.0.

Only those fragments matching the following criteria were considered: a length between 4 and 33 amino acids, at least 2 ion products identified, minimum products per amino acid of 0.2, maximum MH+ error tolerance of 6 ppm, and requiring that the peptide be present in at least two of the three peptide identification runs. Those peptides meeting the filtering criteria were further processed by DynamX 3.0 (Waters Corporation) to calculate the relative amount of deuteration.

Deuterium uptake was obtained by subtracting the centroid mass of the undeuterated form of each peptide from the deuterated form, at each time point, for each condition. Because relative deuterium uptake of individual peptides was compared, no back-exchange correction was performed. The threshold for calling differences in relative deuterium incorporation measurements was set at minimum 0.5 Da.