*3.7. MALDI-TOF MS Analysis*

The characterization changes of *E. coli* K12 bacterial strains were analyzed using a MALDI-TOF MS [11,41–43]. Firstly, 300 μL of ultrapure water and 900 μL of ethanol were mixed, and then 20 mg of the *E. coli* sample was added into the mixture under mild shaking. The sediment was retained after centrifuging (13,000 rpm) for 3 min. Subsequently, 50 μL of CH3CN (acetonitrile) and 50 μL of 70% HCOOH (formic acid) were added. The mixture was centrifuged at 13,000 rpm for 2 min again. The above mixture (0.5 μL) and a DHB matrix solution (2,5-dihydroxybenzoic acid solution, 0.5 μL) were dropped on a

plate and allowed to dry. The experiments of the MALDI-TOF MS were performed on an UltrafleXtreme TOF/TOF operating system equipped with a 355 nm N2 laser. The operating conditions were as follows: positive ion mode, mass range (5–20 kDa), and acceleration voltage (20 kV).
