HepG2 cells were washed twice with pre-cooled PBS and added PBS with 1% protease inhibitors. Cellular proteins were extracted through five cycles of freeze-thaw lysis in liquid nitrogen (5 min freezing) followed by 37 ℃ thawing and the lysates were clarified by centrifugation at 12,000 rpm for 15 min at 4°C. The resulting supernatants were collected for total proteins quantification and the bicinchoninic acid (BCA) assay. A 50 μL reaction system containing 200 μg total protein was aliquoted for further proteomics analysis. Experimental groups received either ALA (treatment) or BSA (control) at a final concentration of 100 μM (n=3 replicates per group). Following thorough mixing, samples were incubated at 37°C for 30 min. Pronase was then added at 0.5% (w/w relative to total protein mass) and incubation continued for an additional 30 min at 37°C. Protein samples (±pronase) were mixed with 5× SDS-PAGE loading buffer and denatured by boiling for 5 min, followed by separation via polyacrylamide gel electrophoresi