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Resuspend the cell pellet in 100 μL ultrapure water, mix well, take 50 μL of the suspension, add 200 μL pre-cooled methanol, vortex at 2500 r/min for 2 min, freeze the sample in liquid nitrogen for 5 min, thaw on ice for 5 min, vortex for 2 min, repeat this freeze-thaw-vortex cycle 3 times, centrifuge at 12,000 r/min, 4 °C for 10 min, transfer 200 μL supernatant to a new tube, store at −20 °C for 30 min, centrifuge again under the same conditions, take 180 μL supernatant, filter through a protein precipitation plate, analyze using UPLC (Waters ACQUITY H-Class D) and MS/MS (QTRAP® 6500+), prepare standard solutions, construct a database from chromatographic peak intensities, perform qualitative analysis using MultiQuant 3.0.3 software, for the remaining 50 μL suspension, repeat the freeze-thaw process 3 times, centrifuge, determine protein concentration by the BCA method, and use protein concentrations of parallel samples to calibrate metabolomics data
