2.4 MS-based proteomics
Natural compounds that capable of covalently modify cysteine residues on
SARS-CoV-2 Mpro, as well as the modification site,
were elucidated using a MS-based chemoproteomic approach. To achieve
this, 30 μM SARS-CoV-2
Mpro was co-incubated with LJ (final concentration,
200 μg/mL) at 37 °C for 3 hours. The
adduct was transferred to 10 kDa centrifugal filter tubes to remove
unbound molecules, followed by washing with water. The sample was
denatured in 100 μL of buffer containing 8 M urea for 1 hour and treated
with dithiothreitol and iodoacetamide to reduce and alkylate cysteine
residues, respectively. The protein was digested with chymotrypsin and
trypsin in 100 µL of 50 mM NH4HCO3 at 37
°C overnight with an enzyme-to-substrate ratio of 1:50 (w/w). And
further desalted using a Solid Phase Extraction Cartridge (MonoSpin C18,
GL Sciences). The eluents were resolved in 40 μL of 0.1% formic acid
after being vacuum-dried for analysis.