Activity-guided native-PAGE electroelution coupled with DIA proteomics uncovers the superoxide dismutase and antioxidant network in mature peanut seeds.

Mature peanut (Arachis hypogaea L.) seeds accumulate abundant superoxide dismutase (SOD), but the high abundance of storage proteins masks trace antioxidant enzymes and prevents accurate profiling of endogenous SOD isoforms by conventional proteomics. Here we report an activity-guided workflow that combines native-PAGE NBT zymography, low-temperature dialysis-bag electroelution and DIA-based nano-LC-MS/MS to enrich native SOD complexes from seed crude extracts. The two SOD-active gel bands, B1 and B2, were excised, embedded in agarose and electroeluted to recover intact proteins before proteomic analysis. Across the crude control (CK) and the two enriched fractions, 1737 non-redundant proteins were annotated, of which 775 (44.6%) were detected only in the SOD-active bands and not in CK, demonstrating efficient enrichment of the hidden proteome. Six SOD-associated proteins were identified: one mitochondrial Mn-SOD, the copper chaperone CCS and four Cu/Zn-SOD isoforms. Quantification showed that the combined abundance of Cu/Zn-SODs exceeded that of Mn-SOD by more than 100-fold, identifying Cu/Zn-SOD as the dominant SOD subtype in mature seeds. Eighteen co-enriched ROS-scavenging enzymes were also detected, and integrative transcriptomics confirmed tissue- and development-specific expression of five of the SOD proteins. This low-cost pipeline with near-quantitative recovery overcomes storage-protein interference and resolves the hidden antioxidant proteome of peanut seeds, providing a general strategy for studying isoenzyme repertoires and redox protein networks in crop seeds.