Data-Independent Acquisition proteomics of protein extracted from kernels collected at fifteen and thirty days post self-pollination of the ubp5 mutant heterozygous (smd/+) and wild-type specimens

protein extraction was performed from kernels collected at fifteen and thirty days post self-pollination of the ubp5 mutant heterozygous (smd/+) and wild-type specimens using phenol. For quantifying protein expression levels, 500μg of dissolved protein underwent reduction by 50 mM DTT (3483-12-3, Sigma-Aldrich) at 37°C for 1 hour. Subsequently, 150 mM iodoacetamide (144-48-9, Sigma-Aldrich) was added to modify the reduced sulfhydryl groups. Following a 30-minute incubation period in darkness at room temperature, the protein sample underwent buffer exchange to 50 mM ammonium bicarbonate (A6141, Sigma-Aldrich) through an ultrafiltration column (UFC501096, Millipore). Enzymatic digestion was carried out using trypsin (V5111, Promega), and the resulting digest underwent purification using C18 Spin Tips (84850, Thermo) followed by elution with 60% acetonitrile and rapid drying under vacuum conditions.