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.