The stained gel bands are washed three times with ddH2O, followed by the addition of 150μL of destaining solution to remove the stain. After four rounds of water washing, the gel bands are washed twice with 300μL each of 25mM ammonium bicarbonate, 50% acetonitrile, and 100% acetonitrile, respectively, until the gel dehydrates and turns white. Then, 50μL of 10mM DTT solution is added and the gel is reduced in a 56℃ water bath for 30 minutes. Once the temperature drops to room temperature, an equal volume of 50 mM IAMAC solution is added and the gel is alkylated in the dark for 15 minutes. The gel bands are then washed twice again with 300μL each of 25mM ammonium bicarbonate, 50% acetonitrile, and 100% acetonitrile, and dehydrated until the gel turns white. Next, 15 to 20μL of proteomics-grade trypsin at a concentration of 0.01 ug/ul is added. After the gel has fully rehydrated and become transparent on ice, 30 to 40μL of a 50 mM NH4HCO3 solution containing 10% ACN is added to cover the gel. The gel is then digested overnight at 37℃ in a water bath. After enzymatic digestion, the supernatant is transferred to a new EP tube. To the remaining gel piece, 100 µL of extraction solution (67% acetonitrile with 2% formic acid) is added, incubated at 37℃ for 30 minutes, followed by sonication for 15 minutes, centrifugation, and the supernatants are combined and concentrated by drying, ready for mass spectrometry analysis.