LC-MS Analysis of ShHTL7 Enzymatic Reactions

For LC-MS analysis: a 200 μL reaction solution (100 mM HEPES, 150 mM NaCl, pH 7.0) containing 10, or 400 μM of tested samples 4a, (R)- and (S)-4a, and 4g-i, and 100 μg of recombinant ShHTL7 or its mutants was incubated at 25 °C for 30 min. Subsequently, the solution was filtered and analyzed by a nanoACQUITY UPLC system, which was directly interfaced with an SYNAPT-G2-Si mass spectrometer produced by Waters Company. For LC-MS/MS analysis, the above chemicals treated ShHTL7 reaction solution was subjected to the SDS-PAGE analysis. The corresponding protein band around 30 KDa was excised, respectively, then digested with trypsin (Promega) in NH₄HCO₃ solution (50 mM) at 37 °C for 24 h^{23 (link),26 (link)–28 (link)}. The covalent modification of C₅H₅O₂/C₆H₆O on the peptide was analyzed by a Thermo-Dionex Ultimate 3000 HPLC system, which was directly interfaced with a Thermo Orbitrap Fusion Lumos mass spectrometer. MS-MS spectra are generated by pLabel software^{53 (link),54 (link)}.