*3.7. Analysis of the Products of AlgM4-Mediated Enzymolysis Using UPLC–QTOF–MS/MS*

The purified AlgM4 (0.25 mg/mL) enzyme was mixed with an equal volume of 1.0% (*w*/*v*) SA, polyM, or polyG; incubated in a water bath at 30 ◦C for 6 h; and then concentrated in vacuo. After high-speed centrifugation, the supernatants were collected and filtered through filter membranes with a 0.22 μm pore size. The filtrates were analyzed using liquid chromatography (LC)-MS. The equipment used in the analysis was a UPLC-QTOF-MS/MS system (Waters Corporation, Milford, MA, USA), which consisted of a UPLC I-Class instrument (Waters Corporation, Singapore) and a XEVO G2-S mass spectrometer (Waters Corporation, Milford, MA, USA). The operating conditions of LC were

as follows: ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm, Waters Corporation, Milford, MA, USA); gradient elution with 0.1% formic acid-water (A) and formic acid-acetonitrile (B); flow rate, 0.5 mL/min; column temperature, 35 ◦C; analytic time, 10 min; injection volume, 1.0 μL. The MS conditions were as follows: ion scan mode, negative-ion ESI mode; scan range, 100–1700 Da; ion source temperature, 100 ◦C; desolvation gas temperature, 400 ◦C; desolvation gas flow rate, 1000 L/h; capillary voltage, 2.5 kV; cone voltage, 40 V; low collision energy, 6 V; high collision energy, 35–50 V; data acquisition software, MassLynx 4.1 SCN 884 (Waters Corporation, Milford, MA, USA); data acquisition mode, MSE.
