3.3 Cysteine modification profiling of SARS-CoV-2
Mpro by LJ
After elucidating the natural compounds in LJ extract, we proceeded to
the next step aiming at identifying those capable of covalently
modifying Mpro and to determine the attachment site.
This was achieved using a chemoproteomic approach based on bottom-up
mass spectrometry. The LJ extract was served as a natural compound
library to screen inhibitors that acting as covalent binders.
Recombinant Mpro was directly incubated with LJ
extract under near-physiological conditions, and subsequently, covalent
protein-inhibitor conjugates were digested and evaluated using nanoflow
LC-MS/MS.
To
get a higher peptide coverage, we performed the method of filter aided
sample preparation
(FASP)34,35. The
results demonstrated that the FASP method’s peptide coverage could reach
88.56%, and more satisfactorily, the coverage of cysteine-containing
peptides was up to 100%. The FASP method involves preparing a denatured
enzyme-inhibitor mixture and then performing enzymatic digestion on the
membranes of 10 kDa ultrafiltration tubes. It should be emphasized that
both the catalytic domain cysteines and the cysteines essential for the
formation of the dimeric form were discovered.
Given that each modification imparts specific fixed mass shifts to the
peptide precursor ion and fragment ions, the mass increase corresponding
to compounds adduction were considered as variable modifications of
cysteine. The localization of chemical modification within the peptide
sequence was determined by MS/MS fragment ion matching. Taking quercetin
as an example, a mass shift of 300.02700 Da attributed to the Michael
adduct of quercetin’s o -quinone form was defined as a variable
modification of cysteine during the database searching step. The MS/MS
spectra of the modified peptides were further manually checked and
verified. Table 1 listed the 22 components in LJ that could
covalently bind on Mpro and their binding sites. The
MS2 spectra of all modified peptides of
Mpro were demonstrated in Fig. S55-S70 . The
catalytic site of SARS-CoV-2 Mpro is characterized by
a cysteine-histidine catalytic dyad
(Cys145 and His41)34. In addition to Cys145, the
cysteine residues near the catalytic site (including
Cys22, Cys44, and Cys85) may also
play crucial roles in enzymatic catalysis or stabilizing this viral
enzyme17,36. Peptide-level analysis reveals that
quercetin and caffeic acid can covalently bind to these cysteine
residues. The enzyme is active only as a dimer. Apigenin and quercetin
were found to covalently bind to the cysteine residue (Cys156) at the
dimerization interface, which may result in loss of enzyme
function37. Modifying the remaining cysteine residues
(such as Cys117 and Cys160) on SARS-CoV-2 Mpro,
however, may have a minor impact on the enzyme’s activity.